Human synthetic single-chain antibodies directed against the common epitope of mutant p53 and their uses

ABSTRACT

Isolated polypeptides, isolated polynucleotides or expression vectors encoding same, viral display vehicles which can be specifically bind an exposed epitope shared by mutant, but not wild type, p53 protein are provided. Also provided are methods of inducing apoptosis and treating cancer as well as diagnosing a p53-related cancer using the isolated polypeptides uncovered by the present invention.

RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.11/887,123 filed on May 8, 2009, which is a National Phase of PCT PatentApplication No. PCT/IL2006/000372 having International filing date ofMar. 23, 2006, which claims the benefit of priority of U.S. ProvisionalPatent Application Nos. 60/698,919 filed on Jul. 14, 2005, and60/664,967 filed on Mar. 25, 2005. The contents of the aboveapplications are all incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention relates to methods of treating cancer usingantibodies directed against p53 mutant proteins.

The tumor suppressor gene p53 inhibits tumor growth primarily viainduction of apoptosis. Mutations in the p53 tumor suppressor gene arethe most common genetic alterations and occur in more than half of allhuman tumors. Approximately 90% of these alterations are missensemutations in the DNA-binding core domain responsible forsequence-specific binding of wild-type p53 protein to target genes. Manyof these mutations cause a common conformational change in the p53protein, which results in the exposure of an epitope that is otherwisehidden inside the wild type p53 molecule.

The involvement of p53 mutants in cancer progression was suggested to beassociated either with trans-dominant suppression of wild-type p53 or awild-type p53-independent oncogenic “gain of function”. As wild-type p53forms a tetramer to exert its tumor suppressor activity, it is generallyaccepted that heteromerization of mutant p53 with wild-type p53 drivesthe wild-type protein into a mutant or otherwise inactive conformationwhich leads to the trans-dominant suppression phenomenon. The “gain offunction” of mutant p53 may be attributed to two mutually possiblemechanisms. One is the abrogation of the tumor suppressor activity ofp53 family members, p63 and p73, that were found to physically interactwith mutant p53, but not with the wild-type p53 protein, and tointerfere with their activity. The second involves the ability of mutantp53 to trans-activate or repress specific genes that mediate the variousoncogenic activities of these mutants. Core domain p53 mutants werefound to trans-activate genes, such as multiple drug resistance (MDR-1),c-myc, proliferating cell nuclear antigen (PCNA), interleukin-6 (IL-6)and epidermal growth factor receptor (EGFR) and early growth receptor(EGR-1), these genes being different from those reactivated by wild typep53.

Given the active role of p53 mutants in promoting tumorigenicity,efforts have been made to inactivate their function or to revert theminto a wild-type phenotype. These include the introduction of secondsite suppressor mutations (e.g., N239Y, N268D and H168R) that can atleast partially restore specific DNA binding to mutant p53. In addition,synthetic peptides derived from the C-terminus of the p53 protein, orthe CDB3, a p53-binding protein (p53BP2) derived compound, were found torestore DNA binding followed by transcriptional trans-activation, aswell as induction of p53 dependent apoptosis to tumor cells. Moreover,low molecular weight compounds, such as CP-31398 and PRIMA-1, were shownto restore wild type conformation, transcriptional trans-activation andto induce apoptosis in cells and in human tumor xenografts carryingmutant p53. However, such peptides and compounds lack the ability todistinguish between the wild-type and mutant form of p53, a propertycrucial for targeted treatment.

Thus, novel anti cancer treatment modalities which specifically target abroad range of p53 mutants and not wild-type p53 proteins are desired

More than 90% of the mutations found in the p53 protein produce aconformational change in the p53 protein which results in the exposureof an epitope, which is otherwise hidden in the hydrophobic core of themolecule. Such an epitope was localized to amino acids 212-217 of thehuman p53 protein (GenBank Accession No. NP_(—)000537) or amino acids209-214 of the mouse p53 protein (GenBank Accession No. NP_(—)035770)and has a sequence of FRHSVV (SEQ ID NO:1). Prior studies describe theisolation of a single-chain scFv mouse antibody prepared from a mouseimmunized with SEQ ID NO:1. This antibody (named ME1), was found to beexpressed in the cytosol of mammalian cells and to bind mutant p53protein but not the wild-type p53 protein with an affinity of 10⁻⁷ M(Govorko D, Cohen G and Solomon B., 2001, J. Immunol. Methods. 258:169-81). However, although this antibody presents a useful tool forclarifying the role of mutant p53 in tumor transformation, due to itsmouse origin and its moderate affinity towards the mutated p53, itstherapeutic application is limited.

There is thus a widely recognized need for, and it would be highlyadvantageous to have, a human derived antibody capable of specificallytargeting with high affinity mutant p53 proteins.

SUMMARY OF THE INVENTION

According to one aspect of the present invention there is provided anisolated polynucleotide comprising a nucleic acid sequence encoding apolypeptide capable of specifically binding an exposed epitope shared byp53 mutant proteins and not by wild type p53 protein, wherein anaffinity of said specific binding is less than 25 nanomolar.

According to another aspect of the present invention there is providedan isolated polynucleotide comprising a nucleic acid sequence encoding aCDR-containing polypeptide comprising at least one CDR selected from thegroup consisting of CDR SEQ ID NOs:8-112.

According to still another aspect of the present invention there isprovided an isolated polypeptide comprising an amino acid sequencecapable of specifically binding an exposed epitope shared by p53 mutantproteins and not by wild type p53 protein, wherein an affinity of thespecific binding is less than 25 nanomolar.

According to an additional aspect of the present invention there isprovided an isolated polypeptide comprising an amino acid sequence of atleast one CDR selected from the group consisting of CDR SEQ IDNOs:8-112.

According to yet another aspect of the present invention there isprovided a pharmaceutical composition comprising as an active ingredientthe isolated polynucleotide and a pharmaceutically acceptable carrier.

According to still an additional aspect of the present invention thereis provided a nucleic acid construct comprising the isolatedpolynucleotide and a promoter for directing an expression of saidisolated polynucleotide in cells.

According to a further aspect of the present invention there is provideda method of inducing apoptosis and/or growth arrest of cancer cells,comprising contacting with or expressing in the cancer cells theisolated polypeptide, thereby inducing apoptosis and/or growth arrest ofthe cancer cells.

According to yet a further aspect of the present invention there isprovided a method of treating a subject suffering from or beingpredisposed to a p53-related cancer, comprising administering to orexpressing in cells of the subject a therapeutically effective amount ofthe isolated polypeptide, thereby treating the p53-related cancer in thesubject.

According to still a further aspect of the present invention there isprovided a method of diagnosing a p53-related cancer in a subjectcomprising: (a) contacting a biological sample of the subject with theisolated polypeptide under conditions suitable for immunocomplexformation which comprises the isolated polypeptide and p53 mutantproteins; and (b) detecting formation of the immunocomplex, therebydiagnosing the cancer in the subject.

According to still a further aspect of the present invention there isprovided a use of the isolated polypeptide for the manufacture of amedicament identified for the treatment of a p53-related cancer.

According to still a further aspect of the present invention there isprovided a use of the isolated polynucleotide for the manufacture of amedicament identified for the treatment of a p53-related cancer.

According to still a further aspect of the present invention there isprovided a use of the nucleic acid construct for the manufacture of amedicament identified for the treatment of a p53-related cancer.

According to still a further aspect of the present invention there isprovided a composition comprising a viral display vehicle expressing ona surface thereof a polypeptide capable of specifically binding anexposed epitope shared by p53 mutant proteins and not by wild type p53protein, wherein an affinity of the specific binding is less than 25nanomolar.

According to still a further aspect of the present invention there isprovided a composition comprising a viral display vehicle expressing ona surface thereof a CDR-containing polypeptide comprising at least oneCDR selected from the group consisting of CDR SEQ ID NOs:8-112.

According to still a further aspect of the present invention there isprovided a pharmaceutical composition comprising as an active ingredientthe viral display vehicle, and a pharmaceutically acceptable carrier.

According to still a further aspect of the present invention there isprovided a method of inducing apoptosis and/or growth arrest of cancercells, comprising contacting with the cancer cells the viral displayvehicle, thereby inducing apoptosis and/or growth arrest of the cancercells.

According to still a further aspect of the present invention there isprovided a method of treating a subject suffering from or beingpredisposed to a p53-related cancer, comprising administering to thesubject a therapeutically effective amount of the viral display vehicle,thereby treating the p53-related cancer in the subject.

According to still a further aspect of the present invention there isprovided a use of the viral display vehicle for the manufacture of amedicament identified for the treatment of a p53-related cancer.

According to further features in preferred embodiments of the inventiondescribed below, the pharmaceutical composition, the epitope is as setforth by SEQ ID NO:1.

According to still further features in the described preferredembodiments the polypeptide comprises at least one CDR selected from thegroup consisting of SEQ ID NOs:39-41, 45-47 and 60-62.

According to still further features in the described preferredembodiments the polypeptide is selected from the group consisting of aFab fragment, an Fv fragment, a single chain antibody, a single domainantibody and an antibody.

According to still further features in the described preferredembodiments the single chain antibody is selected from the groupconsisting of SEQ ID NO:113, SEQ ID NO:114 and SEQ ID NO:115.

According to still further features in the described preferredembodiments the nucleic acid construct further comprising an additionalnucleic acid sequence encoding a nuclear localization signal (NLS) fusedto the isolated polypeptide.

According to still further features in the described preferredembodiments the NLS is set forth by SEQ ID NO:134.

According to still further features in the described preferredembodiments the polynucleotide further comprises an additional nucleicacid sequence encoding a drug.

According to still further features in the described preferredembodiments the polypeptide further comprises an amino acid sequence ofa drug.

According to still further features in the described preferredembodiments the polypeptide is attached to a drug.

According to still further features in the described preferredembodiments the drug is a toxin and/or a chemotherapy drug.

According to still further features in the described preferredembodiments the polynucleotide further comprises an additional nucleicacid sequence encoding a detectable label.

According to still further features in the described preferredembodiments the polypeptide further comprises a detectable label.

According to still further features in the described preferredembodiments the detectable label is biotin and digoxigenin.

According to still further features in the described preferredembodiments the biological sample is selected from the group consistingof blood, lymph node biopsy, bone marrow aspirate and a tissue sample.

According to still further features in the described preferredembodiments each of the Fab fragment, the Fv fragment, the single chainantibody, the single domain antibody and the antibody is humanized.

The present invention successfully addresses the shortcomings of thepresently known configurations by providing antibodies and methods ofinhibiting cell growth and inducing apoptosis by using antibodiesdirected against mutant p53 proteins.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. In case of conflict, the patentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in colorphotograph. Copies of this patent with color photograph(s) will beprovided by the Patent and Trademark Office upon request and payment ofnecessary fee.

The invention is herein described, by way of example only, withreference to the accompanying drawings. With specific reference now tothe drawings in detail, it is stressed that the particulars shown are byway of example and for purposes of illustrative discussion of thepreferred embodiments of the present invention only, and are presentedin the cause of providing what is believed to be the most useful andreadily understood description of the principles and conceptual aspectsof the invention. In this regard, no attempt is made to show structuraldetails of the invention in more detail than is necessary for afundamental understanding of the invention, the description taken withthe drawings making apparent to those skilled in the art how the severalforms of the invention may be embodied in practice.

In the drawings:

FIGS. 1 a-b depict amino acid sequence alignments of the twenty humanscFv clones selected for binding the common epitope of mutant p53(FRHSVV; SEQ ID NO:1). The scFvs are set forth by SEQ ID NOs:113-132.FIG. 1 a depicts amino acids of the variable Heavy chain (V_(H)) (1-119)and FIG. 1 b depicts amino acids of the variable light chain (V_(L))(135-245); twenty human scFvs against the mutant p53 common epitope.Red=identical amino acids, blue=amino acids which have low consensusvalues (more than 50% identical) and black=amino acids which are lessthan 50% identical. CDRs are indicated by a black bar.

FIGS. 2 a-b depict the binding of F2 scFv (also referred to herein as“TAR1”) (FIG. 2 a) and E6 scFv (FIG. 2 b) clones to the FRHSVV (SEQ IDNO:1) epitope. Binding was determined using the BIACore at the indicatedconcentrations of antibodies. The sequences of the VH and VL chains ofthe F2 and E6 scFv clones are shown in FIGS. 1 a-b. RU=resonance units.The binding constant was determined using the software that is suppliedwith the BIAcore instrument (www.Biacore.com).

FIG. 3 is a bar graph depicting the binding of scFv F2 to whole p53molecules as determined using an ELISA. ELISA plates were coated withwild-type p53 or mutant p53-R175H, p53-R248H and p53-R273W whole p53proteins which were prepared from recombinant baculovirus infected sf9insect cells (Hupp T. R and Lane D. P., Curr. Biol. 1994, 4. 865-875),and 250 ng scFv F2 was added. Note the specific binding of the scFv F2to the R175H-mutant p53 molecule as compared with the low binding to thewild type p53.

FIGS. 4 a-j are FACS analyses depicting the effect of the scFv F2 onapoptosis in p53 null human lung carcinoma cells (H1299 p53−/−; FIGS. 4f-j) or in human lung carcinoma cells carrying the hot spot mutationR175H(H1299-R175H; FIGS. 4 a-e). Cells were incubated for 24 hours withscFv-F2 at the following concentrations: 0 nM (untreated cells, FIGS. 4a and f), 250 nM (FIGS. 4 b and g), 500 nM (FIGS. 4 c and h), 1 μM(FIGS. 4 d and i) and 2 μM (FIGS. 4 e and j). Following 24 hours, thecells were fixed with ethanol, stained with Propidium Iodide and thecell cycle profile was determined. Numbers indicate the % of apoptoticcells in each preparation [0.8% (FIG. 4 a), 1.9% (FIG. 4 c), 21.8% (FIG.4 d), 32.2% (FIG. 4 e), 0-0.8% (FIGS. 4 f-j)]. Note the significantincrease in apoptosis in the presence of the scFv-F2 antibody in cellsexpressing the R175H p53 mutation.

FIG. 5 is a bar graph depicting increased susceptibility to drugtreatment of cells expressing the scFv F2 antibody. Human lung carcinomacells carrying the R175H mutation (H1299-R175H) were stably transfectedto express the scFv F2 antibody (H1299-R175H-scFv-F2) and the effect ofthe etoposide and cisplatin drugs on cell survival (by cell counts) wasdetermined 48 hours following drug treatment. Note the increasedsusceptibility of the lung carcinoma cells to low concentrations ofcisplatin (0.5 μg/ml) or etoposide (1 μM) in cells expressing the scFvF2antibody.

FIGS. 6 a-d depict inhibition of colony formation in lung carcinomacells expressing the scFv F2 antibody. H1299-R175H cells were stablytransfected to express the scFv F2 antibody (H1299-R175H-scFv-F2) andthe effect of the intracellular expression of scFv F2 on colonyformation was determined. 500-1000 cell/plate of H1299-R175H cells orH1299-R175H-scFv-F2 cells were seeded on plates containing RPMI culturemedium and allowed to grow for two weeks. FIGS. 6 a-b are photographs ofthe Giemsa-stained colonies present following two weeks in culture. FIG.6 a—H1299-R175H cells; FIG. 6 b—H1299-R175H-scFv-F2 cells. FIGS. 6 c-dare bar graphs depicting the colony number (FIG. 6 c) and the colonyarea (FIG. 6 d) of the H1299-R175H or H1299-R175H-scFv-F2 cellsfollowing two weeks in culture. Note the significant decrease in boththe colony number (FIG. 6 c) and colony area (FIG. 6 d) in cellsexpressing the scFv F2 antibody.

FIG. 7 is a graph depicting the anti tumor effect of scFv F2 on humantumor cell xenografts in mice. Nude mice were subcutaneously injectedwith 5×10⁶ of the lung carcinoma H1299-R175H cells. Following threedays, the mice received an intra-tumor injection of 200 μg scFv F2 orPBS (50 μl) as control, which was repeated every other day during twoweeks. The tumor size was measured following 6, 15, 21, 25, 28, 32, and36 days post inoculation with H1299-R175H cells and the tumor volume wascalculated. The results are presented as relative volume V_(t)/V_(o)where V_(t) is the volume at the indicated day and V_(o) is the volumeat the first scFv F2 injection.

FIGS. 8 a-f depict the effect of the scFv F2 antibody on tumor growth invivo. Mice were subcutaneously injected with human xenografts ofH1299-R175H cells followed by intra-tumor injections as described forFIG. 7. Shown are representative photographs of the PBS-injected mice(FIGS. 8 d-f) as compared with the scFv F2-injected mice (FIGS. 8 a-c).The arrows point to the place of xenograft and tumor formation. Note thepresence of large tumors in the PBS-treated mice (FIGS. 8 a-c) and theabsence of such tumors in the scFv F2 treated mice (FIGS. 8 d-f).

FIG. 9 is a bar graph depicting the binding specificity of scFvs. ELISAwas performed using the F2, A4 and B6 scFvs from the isolated scFvcollection depicted in FIGS. 1 a-b. The binding antigens were BovineSerum Albumin (BSA; lane 5), Streptavidin (lane 6) and the followingpeptides: FRHSVV (SEQ ID NO:1; the common epitope of mutant p53; lane1), amino acids 1-16 of the 13 amyloid peptide DAEFRHDSGYEVHHQK (SEQ IDNO:2; lane 2), amino acids 144-153 of the multiple antigen peptidepresenting the human prion protein (MAP-PrP DYEDRYYRE; SEQ ID NO:3; lane3) and a peptide corresponding to a prostate cancer antigen (PaPILLWQPIPV; SEQ ID NO:4; lane 4). Note the high binding specificity ofthe F2 and A4 antibodies towards the peptide representing the commonepitope of mutant p53 (SEQ ID NO:1).

FIGS. 10 a-d are bar graphs depicting ELISA assays performed onrecombinant p53 wild type or mutant core domains using the TAR1 antibody(FIG. 10 a) or the mAB 1620 specific for wild-type p53 (FIGS. 10 b-d).FIG. 10 a—Binding of TAR1 to mutant p53 R175H core domain (black) andwild-type p53 core domain (gray). Note the high specificity of the TAR1antibody to the p53 mutant R175H core domain (˜0.5 O.D.) as compared tothe p53 wild type core domain (˜0.05 O.D.). FIG. 10 b-Binding of mAb1620 to mutant p53 R175H core domain (black) and wild-type p53 coredomain (gray). Note the high specificity of the mAb 1620 to the p53 wildtype core domain (˜1.2 O.D.) as compared to the mutant p53 R175H coredomain (˜0.3 O.D.). FIGS. 10 c and d—The p53 mutant (R175H) (FIG. 10 c)or wild type (FIG. 10 d) core domains were heated for 30 or 60 minutesat 37° C. in the absence (grey columns) or presence (black columns) of0.4 μM TAR1 and the binding of the mAb 1620 was tested Note thatfollowing heating and in the presence of TAR1, the binding of the mutantp53 to the wild type specific antibody (mAb 1620) is 3 times higher thanin the absence of TAR1 (FIG. 10 c). Also note that binding of mAb 1620to wild type p53 core domain was higher in the presence of TAR1 (FIG. 10d). These results demonstrate that TAR1 induces a conformational changein mutant p53 and stabilizes wild-type p53 conformation.

FIGS. 11 a-b are immunoprecipitation assays depicting the binding of themAb DO-12 mAb, a p53-mutant specific antibody, to wild type or R175Hmutant p53. FIG. 11 a—The p53 mutant (R175H; lanes 1 and 3) or wild-type(WT; lanes 2 and 4) core domains were subjected to immunoprecipitationusing the DO-12 mAb prior to (lanes 1 and 2) or following (lanes 3 and4) incubation for overnight with 80 nM TAR1. Note the high bindingspecificity of the mAb DO-12 to the R175H core domain (lane 1) ascompared to the low binding specificity to the wild type p53 core domain(lane 2). Also note that following overnight incubation with 80 nM TAR1,the binding of the mAb DO-12 to the mutant R175H p53 core domain issignificantly decreased (lane 3), while the binding of the mAb DO-12 tothe wild type p53 core domain is not affected (lane 4). FIG. 11 b—H1299cells expressing mutant p53 R175H were treated over night with 1 μM TAR1and extracts were immunoprecipitated with the mAb 1620 (a p53 wildtype-specific antibody). Note the significant increase, in a TAR1dose-dependent manner, in the binding of mAb 1620 to protein extracts ofcells expressing the mutant p53, demonstrating that TAR1 is capable ofrestoring the wild type p53 conformation in a p53 R175H mutant in vivo.

FIG. 12 is a Circular dichroism analysis depicting the spectra of wildtype or R175H mutant p53 core domains in the presence of TAR1 antibody.Circular dichroism measurements were taken simultaneously for eitherwild-type p53 or mutant p53 R175H core domains and TAR1 separately(TAR1/WT; TAR1/R175H) or as an immunocomplex (TAR1+WT complex;TAR1+R175H complex). Note that binding of TAR1 caused a shift in thespectrum of mutant p53 while almost no difference was observed in thespectrum of the wild-type core domain indicating a conformational changein the complex of TAR1 and mutant p53. Also note that the spectra ofboth TAR1 complexes, with wild-type p53 and with mutant p53, are verysimilar indicating conformational similarity.

FIGS. 13 a-h are Western blot analyses of H1299 cells expressing R175Hmutant p53 extracts probed with anti p21 (FIG. 13 a), MDM2 (FIG. 13 b),Egr1 (FIG. 13 c), Bax (FIG. 13 d) and tubulin (FIGS. 13 e-h) antibodiesdemonstrating the effect of TAR1 treatment on the transcriptionaltransactivation of mutant p53 (R175H). H1299 cells stably expressingmutant p53 were treated for 24 hours with TAR1 at a final concentrationof 0.5 μM (lane 2) or 1 μM (lane 3), or were remained untreated (lane1), following which protein samples were prepared and subjected toWestern blot analyses using the following antibodies: anti p21 (SantaCruz Biotechnology, Inc., dilution 1:1000, MDM2 (was a gift from M.Oren, hybridoma supernatant diluted 1:40), Egr1 (Santa CruzBiotechnology, Inc., dilution 1:500), Bax (Abcam Laboratories, Ltd, UK,dilution 1:1000), tubulin (Sigma, dilution 1:2500). The expression levelof tubulin served as loading control. Note the concentration-dependentincrease in the expression level of p21, MDM2 and Bax following TAR1treatment, demonstrating that TAR1 is capable of restoring thetranscriptional transactivation function to mutant p53. In contrast,note that TAR1 treatment resulted in a decrease in the expression levelof Egr1, demonstrating that TAR1 abrogates the gain of function activityof the p53 mutant protein.

FIG. 14 is a bar graph depicting the quantification of the Western blotanalyses shown in FIGS. 13 a-h. The expression level of each protein inthe treated or untreated cells was normalized by the expression level oftubulin. White bars—untreated cells; grey bars—cells treated for 24hours with 0.5 μM TAR1; black bars—cells treated for 24 hours with 1 μMTAR1.

FIGS. 15 a-h are fluorescence activated cell sorting (FACS) analysesdepicting the effect of TAR1 (scFv-F2) on apoptosis in p53 null H1299human lung carcinoma cells (FIGS. 15 c-d), H1299-R175H cells expressingmutant p53 (FIGS. 15 a-b) or HCT116 colon cancer cells expressing wildtype p53 (FIGS. 15 g-h) or mutant R175H p53 (FIGS. 15 e-f) Cells wereincubated for 24 hours with 1 μM TAR1 (FIGS. 15 b, d, f and h) or wereremained untreated (FIGS. 15 a, c, e and g), following which the cellswere fixed with ethanol, stained with Propidium Iodide and their cellcycle profile was determined. Note the significant increase in apoptosis(Ap fraction) in the presence of the TAR1 antibody in cells expressingthe R175H p53 mutation (FIGS. 15 b and f) as compared to cancer cellswith null p53 or cells expressing the wild-type p53 protein (FIGS. 15 dand h).

FIGS. 16 a-d are TdT-mediated dUTP nick-end labeling (TUNEL) of H1299cells null for p53 (FIGS. 16 c-d) or H1299 cells expressing mutant p53R175H (FIGS. 16 a-b) in the absence (FIGS. 16 a and c) or presence(FIGS. 16 b and d) of 1 μM TAR1. H1299 cells stably transfected toexpress mutant p53 (R175H; FIGS. 16 a-b) or untransfected (FIGS. 16 c-d)were treated for 24 hours with TAR1 (at a final concentration of 1 μM)(FIGS. 16 b and d) or remained untreated (FIGS. 16 a and c) and theeffect of treatment was determined using the TUNEL assay (R&D Systems,Inc).

FIGS. 17 a-r are FACS analyses depicting the effect of TAR1 (scFv-F2) onapoptosis in nine cell lines endogenously expressing different p53mutations. LAN1 (FIGS. 17 a-b), T47D (FIGS. 17 c-d), SKBR3 (FIGS. 17e-f), MCF7 (FIGS. 17 g-h), KM12-C (FIGS. 17 i-j), SW480 (FIGS. 17 k-1),PANC1 (FIGS. 17 m-n), Colon 320 (FIGS. 17 o-p) and MDA231 (FIGS. 17 q-r)cancerous cell lines were treated for 24 hours with 1 μM TAR1 (FIGS. 17b, d, f, h, j, l, n, p and r) or remained untreated (FIGS. 17 a, c, e,g, i, l, m, o and q), following which the cells were subjected toPropidium Iodide FACS analysis as described in FIGS. 15 a-h. Thepercentages of cells in the sub-G1 fraction are indicated. Note thesignificant increase in apoptosis following treatment with the TAR1antibody.

FIGS. 18 a-b are schematic illustrations despicting the construction ofa phage including the PEP internalization peptide. FIG. 18 a—Schematicpresentation of phage PEP cloning into the fd-Tet f88-4 vector (Modifiedfrom Chen, L., et al., 2004, Chem. & Biol. 11, 1081-1091). Also shown isthe sequence of the PEP peptide (EFGACRGDCLGA; SEQ ID NO:136); FIG. 18b—The fd-Tet phage f88-4 vector displaying PEP peptide fused to ˜150copies of the pVIII by cloning into HindIII and PstI sites of therecombinant copy of pVIII gene under the regulation of tac promoter.Note that in order to direct the phage to deliver the anti-p53 antibodyof the present invention (e.g., TAR1) into the nucleus, a nuclearlocalization signal (NLS) peptide (SEQ ID NO:134) can be fused to theantibody and this fusion protein can be cloned upstream of the phageprotein III (FIG. 18 b) to produce a phage that displays TAR1-NLS on itsprotein III.

FIGS. 19 a-c are confocal microscope images depicting immunofluorescenceanalyses of phage PEP in CHO cells and demonstrating thatinternalization of phage is dependent on phage concentration. CHO cellswere incubated for 48 hours with membrane red molecular dye (CM-DiImolecular probe) in the presence of the indicated concentrations ofphage PEP (10¹¹—FIG. 19 a; 10⁹—FIG. 19 b; 10⁶—FIG. 19 c; phage perwell). Internalized phage particles (in green) were detected with mouseanti M13 antibody followed by goat anti mouse cy2 conjugated antibody.Cells were visualized using confocal microscopy. Note that theinternalization of the phage PEP is concentration dependent; the numberof phages detected inside the cells is in direct correlation to theconcentration of phages in the medium, as judged by higher intensity ofgreen labeling inside the cell.

FIGS. 20 a-w are confocal microscope images depicting immunofluorescenceanalyses of phage PEP in CHO cells and demonstrating the penetration ofthe phage into the mammalian CHO cells. Shown are a confocal series ofin depth cellular slices of CHO cells which were incubated for 48 hourswith membrane molecular dye and phage PEP at a final concentration of10¹¹ phage per well. Internalized phage particles were detected withmouse anti M13 antibody followed by goat anti mouse cy2 conjugatedantibody (in green). Membrane was visualized with CM-DiI molecular probe(in red).

FIG. 21 is a bar graph depicting MTT analysis of CHO cells followingincubation with phage PEP. CHO cells were incubated for 48 hours with10⁹ pfu of phage PEP, wild type (W.T.) phage, Timirosal (a control forcell killing) and PBS (cells only) and the cell viability was measuredby the MTT analysis. Error bars represent standard deviation valuescalculated from three independent experiments, with four repeats ineach. Note that the phage PEP is non toxic to CHO cells.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is of isolated polypeptides, isolatedpolynucleotides or expression vectors encoding same which canspecifically bind an exposed epitope shared by mutant, but not wildtype, p53 protein. Specifically, the present invention can be used toinduce apoptosis and treat a p53-related cancer. In addition, thepresent invention can be used to diagnose a p53-related cancer in asubject.

The principles and operation of the isolated polypeptide capable ofbinding the exposed epitope shared by mutant p53 proteins according tothe present invention may be better understood with reference to thedrawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not limited in its applicationto the details set forth in the following description or exemplified bythe Examples. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for the purposeof description and should not be regarded as limiting.

The tumor suppressor gene p53 inhibits tumor growth primarily viainduction of apoptosis. The involvement of p53 mutants in cancerprogression was suggested to be associated with either trans-dominantsuppression of wild-type p53 or wild-type p53-independent oncogenic“gain of function”. Given the active role of p53 mutants in promotingtumorigenicity, efforts have been made to inactivate their function orto revert them into a wild-type phenotype. These include theintroduction of second site suppressor mutations, synthetic peptidesderived from the C-terminus of the p53 protein, or the CDB3 derivedcompound and low molecular weight compounds that were shown to restorewild type conformation, transcriptional trans-activation and to induceapoptosis in cells and in human tumor xenografts carrying mutant p53.However, such peptides and compounds lack the ability to distinguishbetween the wild-type and mutant form of p53, a property crucial fortargeted treatment.

Previous studies were aimed at developing novel anti cancertreatment/diagnostic modalities which specifically target/recognize abroad range of p53 mutants and not the wild-type p53 protein. Forexample, Gannon J V, and co-workers generated a mouse monoclonalantibody [PAb-240, EMBO J. 1990 9(5):1595-602] directed against thecommon epitope of p53 mutant proteins. However, attempts to produce asingle chain antibody from this hybridoma clone failed thus limiting thetherapeutic application of such antibody. Indeed use of this antibodywas suggested only for diagnostic applications. Recently, a single-chainFv (scFv) mouse antibody (ME1) was isolated and was found capable ofbinding exclusively mutant p53 proteins and not the wild type p53protein with an affinity of 10⁻⁷ M (Govorko D, Cohen G and Solomon B.,2001, J. Immunol. Methods. 258: 169-81). However, although this antibodypresents a useful tool for clarifying the role of mutant p53 in tumortransformation, due to its mouse origin and its moderate affinitytowards the mutated p53, its therapeutic application is limited.

While reducing the present invention to practice, the present inventorshave isolated scFvs from a human synthetic combinatorial library[Azriel-Rosenfeld R, et al., 2004, J. Mol. Biol. 335(1): 177-92] whichare capable of specifically binding an epitope shared by mutant p53proteins, but not wild type p53, with a binding affinity of less than 25nanomolar (nM).

As is shown in FIGS. 2 a-b, 3 and 9 and is described in Examples 1 and 2of the Examples section which follows, the F2 (TAR1) and E6 scFvsexhibited an affinity of 1.1×10⁻⁸ and 4.6×10⁻¹⁴ M, respectively towardsthe common epitope (SEQ ID NO:1) shared by mutant, but not wild type,p53 protein. In addition, BIAcore analysis of the A6 scFv revealed abinding constant of 2.3×10⁻⁸ M towards the common epitope (SEQ ID NO:1)(data not shown). Similarly, the F2 scFv (TAR1) exhibited high affinitytowards whole p53 proteins expressing severe (R175H) or intermediate(R248W) conformational mutations but not towards the wild type p53protein. In addition, the TAR1 antibody was shown capable of restoringwild type conformation to mutant p53 core domains (FIGS. 10 a-d, 11 a-band 12, Example 5 of the Examples section which follows) and thetranscriptional transactivation function of wild type p53 (e.g.,activation of wild-type specific target genes such as p21, MDM2 and Bax)while preventing the transcriptional activation of mutant P53 (e.g.,downregulation of Egr1) (FIGS. 13 a-h, 14 and Example 6 of the Examplessection which follows). Such antibodies administered as a polypeptide,displayed on phage or expressed as intrabodies can thus be used for theeffective treatment of p53-related cancer.

Thus, according to one aspect of the present invention there is providedan isolated polypeptide comprising an amino acid sequence capable ofspecifically binding an exposed epitope shared by p53 mutant proteinsand not by wild type p53 protein, wherein an affinity of the specificbinding is less than 25 nM. As used herein the phrase “p53 protein”refers to the TP53 tumor protein p53, a nuclear protein which plays anessential role in the regulation of cell cycle, specifically in thetransition from G0 to G1. p53 is a DNA-binding protein containingDNA-binding, oligomerization and transcription activation domains. p53has been cloned from a variety of sources including, but not limited to,human [GenBank Accession No. NP_(—)000537 (protein) and NM_(—)000546(mRNA)], mouse [GenBank Accession No. NP_(—)035770 (protein) andNM_(—)011640 (mRNA)], rat [GenBank Accession No. NP_(—)112251 (protein)and NM_(—)030989 (mRNA)] and Zebrafish [GenBank Accession No.NP_(—)571402 (protein) and NM_(—)131327 (mRNA)] and the coding sequencesof p53 proteins are available via, for example, the NCBI web site(http://www.ncbi.nlm.nih.gov).

The phrase “exposed epitope” refers to any antigenic determinant whichin the wild type p53 protein is hidden within a hydrophobic core, but inp53 mutants which undergo intermediate or severe conformational changesis exposed to the outer surface of the protein. For example, such anexposed epitope can be the FRHSVV (SEQ ID NO:1) which is exposed in p53proteins carrying the R248W and R175H, intermediate and severemutations, but is hidden in the wild type p53 protein.

The phrase “affinity of the specific binding” refers to the bindingaffinity of the isolated polypeptide of the present invention to theexposed epitope shared by p53 mutant proteins. Such affinity can bemeasured using methods known in the arts [e.g., the BIAcore system(Biacore AB, Uppsala, Sweden), scatchard plot analysis] in which thedissociation (K_(d)) or binding K_(a)) constants can be calculated.According to this aspect of the present invention the affinity of thepolypeptide of the present invention is characterized with adissociation constant of less than 25 nM, preferably, less than 12 nM,preferably less than 1 nM, preferably less than 0.1 nM and even morepreferably less than 0.01 nM.

Antibodies of this aspect of the present invention are capable ofneutralizing activity of mutant p53 such as preventing itstranscriptional activation activity (as demonstrated in FIGS. 13-14 andExample 6 of the Examples section which follows).

The isolated polypeptide of the present invention can be any synthetic,natural occurring or recombinantly expressed polypeptide which iscapable of binding the exposed epitope shared by p53 mutant proteins butnot the wild type p53 protein. Such a polypeptide is preferably anantibody or an antibody fragment.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof, such as Fab, F(ab′)2, Fv orsingle domain molecules such as VH and VL to an epitope of an antigen atleast partially generated by recombinant DNA technology. Thesefunctional antibody fragments are defined as follows: (1) Fab, thefragment which contains a monovalent antigen-binding fragment of anantibody molecule, can be produced by digestion of whole antibody withthe enzyme papain to yield an intact light chain and a portion of oneheavy chain; (2) Fab′, the fragment of an antibody molecule that can beobtained by treating whole antibody with pepsin, followed by reduction,to yield an intact light chain and a portion of the heavy chain; twoFab′ fragments are obtained per antibody molecule; (3) (Fab′)2, thefragment of the antibody that can be obtained by treating whole antibodywith the enzyme pepsin without subsequent reduction; F(ab′)2 is a dimerof two Fab′ fragments held together by two disulfide bonds; (4) Fv,defined as a genetically engineered fragment containing the variableregion of the light chain and the variable region of the heavy chainexpressed as two chains; (5) Single chain antibody (“SCA”), agenetically engineered molecule containing the variable region of thelight chain and the variable region of the heavy chain, linked by asuitable polypeptide linker as a genetically fused single chain molecule(scFv); and (6) Single domain antibodies are composed of a single VH orVL domains which exhibit sufficient affinity to the antigen.

The term “antibody” as used herein is not only inclusive of antibodiesgenerated by immunization and recombinant phage display techniques, butalso includes any polypeptide which is generated to include at least onecomplementary-determining region (CDR) which is capable of specificallybinding the exposed epitope shared by mutant p53 but not wild type p53protein. Thus, the antibody of the present invention can be expressed(as is further described hereinbelow) from a polynucleotide sequenceincluding a coding sequence of at least one CDR of an antibody.

To increase avidity of the antibody towards the exposed epitope sharedby mutant but not wild type p53, antibodies of the present invention arepreferably at least bivalent. Such antibodies can be cloned into an IgGsubtype (which is bivalent), an IgM (which is penta-valent) or selectedof such subtypes using methods known in the arts. Alternatively, theaffinity of single chain variable fragments (scFvs) can be increased bytetramerization on streptavidin following site-specific biotinylation bythe enzyme BirA, essentially as described in Cloutier S M, et al., 2000,Mol. Immunol. 37(17): 1067-77.

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988, incorporated herein by reference).

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ormammalian cells (e.g. Chinese hamster ovary cell culture or otherprotein expression systems) of DNA encoding the fragment. Antibodyfragments can be obtained by pepsin or papain digestion of wholeantibodies by conventional methods. For example, antibody fragments canbe produced by enzymatic cleavage of antibodies with pepsin to provide a5S fragment denoted F(ab′)2. This fragment can be further cleaved usinga thiol reducing agent, and optionally a blocking group for thesulfhydryl groups resulting from cleavage of disulfide linkages, toproduce 3.5S Fab′ monovalent fragments. Alternatively, an enzymaticcleavage using pepsin produces two monovalent Fab′ fragments and an Fcfragment directly. These methods are described, for example, byGoldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and referencescontained therein, which patents are hereby incorporated by reference intheir entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)].Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

Fv fragments comprise an association of VH and VL chains. Thisassociation may be noncovalent, as described in Inbar et al. [Proc.Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise VH and VL chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (scFv) are prepared byconstructing a structural gene comprising DNA sequences encoding the VHand VL domains connected by an oligonucleotide. The structural gene isinserted into an expression vector, which is subsequently introducedinto a host cell such as E. coli. The recombinant host cells synthesizea single polypeptide chain with a linker peptide bridging the two Vdomains. Methods of producing scFvs are described, for example, byWhitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); andU.S. Pat. No. 4,946,778, which is hereby incorporated by reference inits entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementary-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry[Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′)₂ or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the framework regions (FR) are those of a humanimmunoglobulin consensus sequence. The humanized antibody optimally alsowill comprise at least a portion of an immunoglobulin constant region(Fc), typically that of a human immunoglobulin [Jones et al., Nature,321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991). Theconstruction of large human synthetic single-chain Fv antibody librarieswhere in vivo formed CDRs are shuffled combinatorially ontogermline-derived human variable-region frameworks are also available[Azriel-Rosenfeld R, et al., 2004. A human synthetic combinatoriallibrary of arrayable single-chain antibodies based on shuffling in vivoformed CDRs into general framework regions. J. Mol. Biol. 335(1):177-92]. Similarly, human antibodies can be made by introduction ofhuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13, 65-93 (1995).

It will be appreciated that targeting of particular compartment withinthe cell can be achieved using intracellular antibodies (also known as“intrabodies”). These are essentially SCA to which intracellularlocalization signals have been added (e.g., ER, mitochondrial, nuclear,cytoplasmic). This technology has been successfully applied in the art(for review, see Richardson and Marasco, 1995, TIBTECH vol. 13).Intrabodies have been shown to virtually eliminate the expression ofotherwise abundant cell surface receptors and to inhibit a proteinfunction within a cell (See, for example, Richardson et al., 1995, Proc.Natl. Acad. Sci. USA 92: 3137-3141; Deshane et al., 1994, Gene Ther. 1:332-337; Marasco et al., 1998 Human Gene Ther 9: 1627-42; Shaheen etal., 1996 J. Virol. 70: 3392-400; Werge, T. M. et al., 1990, FEBSLetters 274:193-198; Carlson, J. R. 1993 Proc. Natl. Acad. Sci. USA90:7427-7428; Biocca, S. et al., 1994, Bio/Technology 12: 396-399; Chen,S-Y. et al., 1994, Human Gene Therapy 5:595-601; Duan, L et al., 1994,Proc. Natl. Acad. Sci. USA 91:5075-5079; Chen, S-Y. et al., 1994, Proc.Natl. Acad. Sci. USA 91:5932-5936; Beerli, R. R. et al., 1994, J. Biol.Chem. 269:23931-23936; Mhashilkar, A. M. et al., 1995, EMBO J.14:1542-1551; PCT Publication No. WO 94/02610 by Marasco et al.; and PCTPublication No. WO 95/03832 by Duan et al.).

To prepare an intracellular antibody expression vector, the cDNAencoding the antibody light and heavy chains specific for the targetprotein of interest are isolated, typically from a hybridoma thatsecretes a monoclonal antibody specific for the marker. Hybridomassecreting anti-marker monoclonal antibodies, or recombinant monoclonalantibodies, can be prepared using methods known in the art. Once amonoclonal antibody specific for the marker protein is identified (e.g.,either a hybridoma-derived monoclonal antibody or a recombinant antibodyfrom a combinatorial library), DNAs encoding the light and heavy chainsof the monoclonal antibody are isolated by standard molecular biologytechniques. For hybridoma derived antibodies, light and heavy chaincDNAs can be obtained, for example, by PCR amplification or cDNA libraryscreening. For recombinant antibodies, such as from a phage displaylibrary, cDNA encoding the light and heavy chains can be recovered fromthe display package (e.g., phage) isolated during the library screeningprocess and the nucleotide sequences of antibody light and heavy chaingenes are determined. For example, many such sequences are disclosed inKabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242 and in the “Vbase” human germline sequencedatabase. Once obtained, the antibody light and heavy chain sequencesare cloned into a recombinant expression vector using standard methods.

For cytoplasmic expression of the light and heavy chains, the nucleotidesequences encoding the hydrophobic leaders of the light and heavy chainsare removed. To direct the expression of an antibody to the cell nuclei,a nuclear localization signal coding sequence (e.g., DPKKKRKV; SEQ IDNO:134) is preferably ligated to a nucleic acid construct encoding theantibody, preferably, downstream of the coding sequence of the antibody.A non-limiting example of such a configuration is provided under theMaterials and Experimental Methods of the Examples section whichfollows. An intracellular antibody expression vector can encode anintracellular antibody in one of several different forms. For example,in one embodiment, the vector encodes full-length antibody light andheavy chains such that a full-length antibody is expressedintracellularly. In another embodiment, the vector encodes a full-lengthlight chain but only the VH/CH1 region of the heavy chain such that aFab fragment is expressed intracellularly. In another embodiment, thevector encodes a single chain antibody (scFv) wherein the variableregions of the light and heavy chains are linked by a flexible peptidelinker [e.g., (Gly₄Ser)₃ and expressed as a single chain molecule. Toinhibit marker activity in a cell, the expression vector encoding theintracellular antibody is introduced into the cell by standardtransfection methods, as discussed hereinbefore.

According to preferred embodiments of this aspect of the presentinvention the polypeptide of the present invention is a recombinantpolypeptide comprising at least one CDR sequence as set forth by SEQ IDNOs:8-112. Examples include, but are not limited to the F2 scFv (SEQ IDNO:113), the E6 scFv (SEQ ID NO:114) and the A6 scFv (SEQ ID NO:115and/or any IgG clone including at least one CDR or scFv sequence.Preferably, the polypeptide of the present invention comprises at leastone CDR as set forth by SEQ ID NOs:39-41, 45-47 and 60-62.

According to preferred embodiments of this aspect of the presentinvention the polypeptide of the present invention can be a Fabfragment, an Fv fragment, a single chain antibody and a single domainantibody. Non-limiting examples of the single chain antibody which canbe used along with the present invention include the F2 scFv (SEQ IDNO:113) and E6 scFv (SEQ ID NO:114) and A6 scFv (SEQ ID NO:115), all ofwhich exhibit high affinity (with a dissociation constant being lessthan 25 nM) towards to exposed epitope shared by p53 mutants by not thewild type p53.

As is shown in Tables 3-5 of the Examples section which follows, thepresent inventors have isolated twenty different scFvs that specificallybind the mutant p53 common epitope (SEQ ID NO:1). These scFvs share anidentical heavy variable region (Table 3) and differ in their lightvariable region (Table 4). Table 5 presents the unique CDRs of thevariable light chains which can specifically bind a p53 epitope sharedby various mutants of the p53 protein but not by the wild type p53protein.

Thus, according to another aspect of the present invention there isprovided an isolated polypeptide comprising at least one CDR selectedfrom the group consisting of CDR SEQ ID NOs:8-112.

As is further described in the Examples section which follows and in thedescription hereinbelow, the isolated polypeptide of the presentinvention can be used in various in vitro, ex vivo and in vivoapplications. To increase the stability, bioavailability, affinity andavidity to the target epitope (i.e., to the exposed epitope shared bymutants but not wild type p53 protein), the present invention can alsoemploy peptides, peptide analogues or mimetics thereof derived from theCDRs of the present invention (e.g., SEQ ID NOs:8-112).

Such peptides, peptide analogues or mimetics thereof are preferablyshort amino acid sequences of at least 4-5 amino acids, preferably atleast 6, more preferably, at least 7, more preferably, in the range of8-20, more preferably in the range of 8-15, even more preferably, in therange of 11-15 amino acids which are derived from at least one CDRs ofthe CDRs set forth by SEQ ID NOs:8-112. The term “peptide” as usedherein encompasses native peptides (either degradation products,synthetically synthesized peptides or recombinant peptides) and asmentioned hereinabove, peptidomimetics (typically, syntheticallysynthesized peptides), as well as peptoids and semipeptoids which arepeptide analogs, which may have, for example, modifications renderingthe peptides more stable while in a body or more capable of penetratinginto cells. Such modifications include, but are not limited to Nterminus modification, C terminus modification, peptide bondmodification, including, but not limited to, CH2-NH, CH2-S, CH2-S═O,O═C—NH, CH2-O, CH2-CH2, S═C—NH, CH═CH or CF═CH, backbone modifications,and residue modification. Methods for preparing peptidomimetic compoundsare well known in the art and are specified, for example, inQuantitative Drug Design, C. A. Ramsden Gd., Chapter 17.2, F. ChoplinPergamon Press (1992), which is incorporated by reference as if fullyset forth herein. Further details in this respect are providedhereinunder.

Peptide bonds (—CO—NH—) within the peptide may be substituted, forexample, by N-methylated bonds (—N(CH3)-CO—), ester bonds(—C(R)H—C—O—O—C(R)—N—), ketomethylen bonds (—CO—CH2-), α-aza bonds(—NH—N(R)—CO—), wherein R is any alkyl, e.g., methyl, carba bonds(—CH2-NH—), hydroxyethylene bonds (—CH(OH)—CH2-), thioamide bonds(—CS—NH—), olefinic double bonds (—CH═CH—), retro amide bonds (—NH—CO—),peptide derivatives (—N(R)—CH2-CO—), wherein R is the “normal” sidechain, naturally presented on the carbon atom.

These modifications can occur at any of the bonds along the peptidechain and even at several (2-3) at the same time.

Natural aromatic amino acids, Trp, Tyr and Phe, may be substituted forsynthetic non-natural acid such as Phenylglycine, TIC, naphthylelanine(Nol), ring-methylated derivatives of Phe, halogenated derivatives ofPhe or o-methyl-Tyr.

In addition to the above, the peptides of the present invention may alsoinclude one or more modified amino acids or one or more non-amino acidmonomers (e.g. fatty acids, complex carbohydrates etc).

As used herein in the specification the term “amino acid” or “aminoacids” is understood to include the 20 naturally occurring amino acids;those amino acids often modified post-translationally in vivo,including, for example, hydroxyproline, phosphoserine andphosphothreonine; and other unusual amino acids including, but notlimited to, 2-aminoadipic acid, hydroxylysine, isodesmosine, nor-valine,nor-leucine and ornithine. Furthermore, the term “amino acid” includesboth D- and L-amino acids.

Tables 1 and 2 below list naturally occurring amino acids (Table 1) andnon-conventional or modified amino acids (Table 2) which can be usedwith the present invention.

TABLE 1 Amino Acid Three-Letter Abbreviation One-letter Symbol alanineAla A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys CGlutammine Gln Q Glutamic Acid Glu E glycine Gly G Histidine His Hisoleucine Iie I leucine Leu L Lysine Lys K Methionine Met Mphenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr Ttryptophan Trp W tyrosine Tyr Y Valine Val V Any amino acid as Xaa Xabove

TABLE 2 Non-conventional Non-conventional amino amino acid Code acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgincarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornthine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-metyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcyclopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-naphthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cyclododeclglycine Ncdod D-α-methylalnine Dnmala N-cyclooctylglycineNcoct D-α-methylarginine Dnmarg N-cyclopropylglycine NcproD-α-methylasparagine Dnmasn N-cycloundecylglycine NcundD-α-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-α-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylleucine Dnmleu N-(3-indolylethyl) glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvaD-N-methyltyrosine Dnmtyr N-methyla-naphthyalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg Lα-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyeethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine NhisD-N-methylleucine Dnmleu N-(3-indolylethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine MncpenN-methylglysine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine mser L-α-methylthreonine Mthr L-α-methylvaline MtrpL-α-methyltyrosine Mtyr L-α-methylleucine Mval L-N- Nmhphe Nnbhmmethylhomophenylalanine N-(N-(2,2-diphenylethyl)N-(N-(3,3-diphenylpropyl) carbamylmethyl-glycine Nnbhmcarbamylmethyl(1)glycine Nnbhe 1-carboxy-1-(2,2- Nmbcdiphenylethylamino)cyclopropane

The peptides of the present invention are preferably utilized in alinear form, although it will be appreciated that in cases wherecyclicization does not severely interfere with peptide characteristics,cyclic forms of the peptide can also be utilized.

The isolated polypeptide of the present invention (e.g., peptideincluding at least one of the CDRs set forth by SEQ ID NOs:8-112) can bebiochemically synthesized using standard solid phase techniques. Thesemethods include exclusive solid phase synthesis, partial solid phasesynthesis methods, fragment condensation and classical solutionsynthesis. These methods are preferably used when the peptide isrelatively short (i.e., 10 kDa) and/or when it cannot be produced byrecombinant techniques (i.e., not encoded by a nucleic acid sequence)and therefore involve different chemistry.

Solid phase peptide synthesis procedures are well known in the art andfurther described by John Morrow Stewart and Janis Dillaha Young, SolidPhase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Synthetic peptides can be purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing.

In cases where large amounts of the peptides of the present inventionare desired, the peptides of the present invention can be generatedusing recombinant techniques such as described by Bitter et al., (1987)Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods inEnzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsuet al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.3:1671-1680; Brogli et al., (1984) Science 224:838-843; Gurley et al.(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp 421-463.

As used herein the term “mimetics” refers to molecular structures, whichserve as substitutes for the peptide of the present invention inspecifically binding the exposed epitope of p53 shared by mutants, butnot wild type p53 protein (Morgan et al. (1989) Ann. Reports Med. Chem.24:243-252 for a review of peptide mimetics).

Peptide mimetics, as used herein, include synthetic structures (knownand yet unknown), which may or may not contain amino acids and/orpeptide bonds, but retain the structural and functional features ofspecifically binding the exposed epitope of p53 shared by mutants, butnot the wild type p53 protein. Types of amino acids which can beutilized to generate mimetics are further described hereinbelow. Theterm, “peptide mimetics” also includes peptoids and oligopeptoids, whichare peptides or oligomers of N-substituted amino acids [Simon et al.(1972) Proc. Natl. Acad. Sci. USA 89:9367-9371]. Further included aspeptide mimetics are peptide libraries, which are collections ofpeptides designed to be of a given amino acid length and representingall conceivable sequences of amino acids corresponding thereto.

Generation of peptide mimetics, as described hereinabove, is effectedusing various approaches, including, for example, display techniques,using a plurality of display vehicles (such as phages, viruses orbacteria) each displaying a short peptide sequence as describedhereinabove. Methods of constructing and screening peptide displaylibraries are well known in the art. Such methods are described in, forexample, Young A C, et al., “The three-dimensional structures of apolysaccharide binding antibody to Cryptococcus neoformans and itscomplex with a peptide from a phage display library: implications forthe identification of peptide mimotopes” J Mol Biol 1997 Dec. 12;274(4):622-34; Giebel L B et al. “Screening of cyclic peptide phagelibraries identifies ligands that bind streptavidin with highaffinities” Biochemistry 1995 Nov. 28; 34(47):15430-5; Davies E L etal., “Selection of specific phage-display antibodies using librariesderived from chicken immunoglobulin genes” J Immunol Methods 1995 Oct.12; 186(1):125-35; Jones C R T al. “Current trends in molecularrecognition and bioseparation” J Chromatogr A 1995 Jul. 14; 707(1):3-22;Deng S J et al. “Basis for selection of improved carbohydrate-bindingsingle-chain antibodies from synthetic gene libraries” Proc Natl AcadSci USA 1995 May 23; 92(11):4992-6; and Deng S J et al. “Selection ofantibody single-chain variable fragments with improved carbohydratebinding by phage display” J Biol Chem 1994 Apr. 1; 269(13):9533-8, whichare incorporated herein by reference.

Peptide mimetics can also be uncovered using computational biology. Forexample, various compounds can be computationally analyzed for theability to specifically bind the exposed epitope of p53 shared bymutants, but not wild type p53 protein using a variety ofthree-dimensional computational tools. Software programs useful fordisplaying three-dimensional structural models, such as RIBBONS (Carson,M., 1997. Methods in Enzymology 277, 25), O (Jones, T A. et al., 1991.Acta Crystallogr. A47, 110), DINO (DINO: Visualizing Structural Biology(2001) http://www.dino3d.org); and QUANTA, INSIGHT, SYBYL, MACROMODE,ICM, MOLMOL, RASMOL and GRASP (reviewed in Kraulis, J., 1991. ApplCrystallogr. 24, 946) can be utilized to model interactions between theexposed epitope of p53 (e.g., SEQ ID NO:1) and prospective peptidemimetics to thereby identify peptides which display the highestprobability of specifically binding the exposed epitope shared bymutants, but not wild type, p53 protein. Computational modeling ofprotein-peptide interactions has been successfully used in rational drugdesign, for further detail, see Lam et al., 1994. Science 263, 380;Wlodawer et al., 1993. Ann Rev Biochem. 62, 543; Appelt, 1993.Perspectives in Drug Discovery and Design 1, 23; Erickson, 1993.Perspectives in Drug Discovery and Design 1, 109, and Mauro M J. et al.,2002. J Clin Oncol. 20, 325-34.

Regardless of the methods employed, peptide mimetics generated using theabove-teachings can be qualified using the assays described in theExamples section which follows (e.g., binding affinity assay).

As is mentioned before, the isolated polypeptide of the presentinvention can be also recombinantly expressed in cells (e.g., mammaliancells, bacterial cells, plant cells, yeast cells) as part of a nucleicacid construct containing a polynucleotide encoding for example, the Fabfragment, the scFV, the complete IgG antibody or any of the CDRs of theisolated polypeptide of the present invention.

To express the recombinant isolated polypeptide of the present inventionin mammalian cells, a polynucleotide sequence encoding at least one CDRsequence as set forth by SEQ ID NOs:8-112 is preferably ligated into anucleic acid construct suitable for mammalian cell expression. Such apolynucleotide can be, for example, the polynucleotide set forth by SEQID NO:113 (for the F2 scFv), SEQ ID NO:114 (for the E6 scFv), SEQ IDNO:115 (for the A6 scFv). The nucleic acid construct includes a promotersequence for directing transcription of the polynucleotide sequence inthe cell in a constitutive or inducible manner.

Constitutive promoters suitable for use with the present invention arepromoter sequences which are active under most environmental conditionsand most types of cells such as the cytomegalovirus (CMV) and Roussarcoma virus (RSV). Inducible promoters suitable for use with thepresent invention include for example the tetracycline-induciblepromoter (Zabala M, et al., Cancer Res. 2004, 64(8): 2799-804).

The nucleic acid construct (also referred to herein as an “expressionvector”) of the present invention includes additional sequences whichrender this vector suitable for replication and integration inprokaryotes, eukaryotes, or preferably both (e.g., shuttle vectors). Inaddition, typical cloning vectors may also contain a transcription andtranslation initiation sequence, transcription and translationterminator and a polyadenylation signal. By way of example, suchconstructs will typically include a 5′ LTR, a tRNA binding site, apackaging signal, an origin of second-strand DNA synthesis, and a 3′ LTRor a portion thereof.

Eukaryotic promoters typically contain two types of recognitionsequences, the TATA box and upstream promoter elements. The TATA box,located 25-30 base pairs upstream of the transcription initiation site,is thought to be involved in directing RNA polymerase to begin RNAsynthesis. The other upstream promoter elements determine the rate atwhich transcription is initiated.

Preferably, the promoter utilized by the nucleic acid construct of thepresent invention is active in the specific cell population transformed.Examples of cell type-specific and/or tissue-specific promoters includepromoters such as albumin that is liver specific [Pinkert et al., (1987)Genes Dev. 1:268-277], lymphoid specific promoters [Calame et al.,(1988) Adv. Immunol. 43:235-275]; in particular promoters of T-cellreceptors [Winoto et al., (1989) EMBO J. 8:729-733] and immunoglobulins;[Banerji et al. (1983) Cell 33729-740], neuron-specific promoters suchas the neurofilament promoter [Byrne et al. (1989) Proc. Natl. Acad.Sci. USA 86:5473-5477], pancreas-specific promoters [Edlunch et al.(1985) Science 230:912-916] or mammary gland-specific promoters such asthe milk whey promoter (U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166).

Enhancer elements can stimulate transcription up to 1,000 fold fromlinked homologous or heterologous promoters. Enhancers are active whenplaced downstream or upstream from the transcription initiation site.Many enhancer elements derived from viruses have a broad host range andare active in a variety of tissues. For example, the SV40 early geneenhancer is suitable for many cell types. Other enhancer/promotercombinations that are suitable for the present invention include thosederived from polyoma virus, human or murine cytomegalovirus (CMV), thelong term repeat from various retroviruses such as murine leukemiavirus, murine or Rous sarcoma virus and HIV. See, Enhancers andEukaryotic Expression, Cold Spring Harbor Press, Cold Spring Harbor,N.Y. 1983, which is incorporated herein by reference.

In the construction of the expression vector, the promoter is preferablypositioned approximately the same distance from the heterologoustranscription start site as it is from the transcription start site inits natural setting. As is known in the art, however, some variation inthis distance can be accommodated without loss of promoter function.

Polyadenylation sequences can also be added to the expression vector inorder to increase the efficiency of recombinant isolated antibody mRNAtranslation. Two distinct sequence elements are required for accurateand efficient polyadenylation: GU or U rich sequences located downstreamfrom the polyadenylation site and a highly conserved sequence of sixnucleotides, AAUAAA, located 11-30 nucleotides upstream. Termination andpolyadenylation signals that are suitable for the present inventioninclude those derived from SV40.

In addition to the elements already described, the expression vector ofthe present invention may typically contain other specialized elementsintended to increase the level of expression of cloned nucleic acids orto facilitate the identification of cells that carry the recombinantDNA. For example, a number of animal viruses contain DNA sequences thatpromote the extra chromosomal replication of the viral genome inpermissive cell types. Plasmids bearing these viral replicons arereplicated episomally as long as the appropriate factors are provided bygenes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

The expression vector of the present invention can further includeadditional polynucleotide sequences that allow, for example, thetranslation of several proteins from a single mRNA such as an internalribosome entry site (IRES) and sequences for genomic integration of thepromoter-chimeric polypeptide.

Examples for mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp2O5. Other exemplary vectors include pMSG, pAV009/A⁺, pMT010/A⁺,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

As described above, viruses are very specialized infectious agents thathave evolved, in many cases, to elude host defense mechanisms.Typically, viruses infect and propagate in specific cell types. Thetargeting specificity of viral vectors utilizes its natural specificityto specifically target predetermined cell types and thereby introduce arecombinant gene into the infected cell. Thus, the type of vector usedby the present invention will depend on the cell type transformed. Theability to select suitable vectors according to the cell typetransformed is well within the capabilities of the ordinary skilledartisan and as such no general description of selection consideration isprovided herein. For example, bone marrow cells can be targeted usingthe human T cell leukemia virus type I (HTLV-I) and kidney cells may betargeted using the heterologous promoter present in the baculovirusAutographa californica nucleopolyhedrovirus (AcMNPV) as described inLiang C Y et al., 2004 (Arch Virol. 149: 51-60).

Recombinant viral vectors are useful for in vivo expression of theisolated polypeptide of the present invention since they offeradvantages such as lateral infection and targeting specificity. Lateralinfection is inherent in the life cycle of, for example, retrovirus andis the process by which a single infected cell produces many progenyvirions that bud off and infect neighboring cells. The result is that alarge area becomes rapidly infected, most of which was not initiallyinfected by the original viral particles. This is in contrast tovertical-type of infection in which the infectious agent spreads onlythrough daughter progeny. Viral vectors can also be produced that areunable to spread laterally. This characteristic can be useful if thedesired purpose is to introduce a specified gene into only a localizednumber of targeted cells.

Various methods can be used to introduce the expression vector of thepresent invention into cells. Such methods are generally described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringsHarbor Laboratory, New York (1989, 1992), in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.(1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995),Vectors: A Survey of Molecular Cloning Vectors and Their Uses,Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4(6): 504-512, 1986] and include, for example, stable or transienttransfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Other than containing the necessary elements for the transcription andtranslation of the inserted coding sequence, the expression construct ofthe present invention can also include sequences engineered to enhancestability, production, purification, secretion, yield or toxicity of theexpressed peptide.

For secretion of the isolated polypeptide of the present invention thenucleic acid construct of the present invention typically includes asignal sequence for secretion of the peptide from a host cell in whichit is placed. In addition, the expression of a fusion protein or acleavable fusion protein comprising Met variant of the present inventionand a heterologous protein can be engineered. Such a fusion protein canbe designed so that the fusion protein can be readily isolated byaffinity chromatography; e.g., by immobilization on a column specificfor the heterologous protein. Where a cleavage site is engineeredbetween the Met moiety and the heterologous protein, the Met moiety canbe released from the chromatographic column by treatment with anappropriate enzyme or agent that disrupts the cleavage site [e.g., seeBooth et al. (1988) Immunol. Lett. 19:65-70; and Gardella et al., (1990)J. Biol. Chem. 265:15854-15859].

It will be appreciated that a variety of prokaryotic or eukaryotic cellscan be used as host-expression systems to express the isolatedpolypeptide of the present invention. These include, but are not limitedto, microorganisms, such as bacteria transformed with a recombinantbacteriophage DNA, plasmid DNA or cosmid DNA expression vectorcontaining the coding sequence; yeast transformed with recombinant yeastexpression vectors containing the coding sequence; plant cell systemsinfected with recombinant virus expression vectors (e.g., cauliflowermosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed withrecombinant plasmid expression vectors, such as Ti plasmid, containingthe coding sequence. Mammalian expression systems can also be used toexpress the polypeptides of the present invention.

Examples of bacterial constructs include the pET series of E. coliexpression vectors [Studier et al. (1990) Methods in Enzymol.185:60-89).

In yeast, a number of vectors containing constitutive or induciblepromoters can be used, as disclosed in U.S. Pat. No. 5,932,447.Alternatively, vectors can be used which promote integration of foreignDNA sequences into the yeast chromosome.

Other expression systems such as insects, plants and mammalian host cellsystems are well known in the art and can also be used by the presentinvention.

Recovery of the recombinant polypeptide of the present invention iseffected following an appropriate time in culture. The phrase“recovering the recombinant polypeptide” refers to collecting the wholegrowth (e.g., fermentation) medium containing the polypeptide and neednot imply additional steps of separation or purification. Notwithstanding the above, polypeptides of the present invention can bepurified using a variety of standard protein purification techniques,such as, but not limited to, affinity chromatography, ion exchangechromatography, filtration, electrophoresis, hydrophobic interactionchromatography, gel filtration chromatography, reverse phasechromatography, chromatofocusing and differential solubilization.

It will be appreciated that the isolated polypeptide of the presentinvention (which acts as an anti p53 antibody) can be also expressed inthe target cells which express mutant p53 protein (e.g., cancer cellsharboring a severe p53 mutation such as R175H) to thereby form anintracellular antibody as described hereinabove. This is of particularimportance especially in the case of p53 which is a nuclear protein andthus is relatively more “resistance” to conventional antibody therapy.As is shown in FIGS. 4 a-j, 15 a-h and 16 a-d and is described inExample 3 of the Examples section which follows, the F2 scFv antibody(TAR1) was capable of inducing apoptosis in human lung carcinoma orhuman colon cancer cells specifically expressing the mutant R175H p53protein (with the exposed epitope set forth by SEQ ID NO:1). Inaddition, treatment of cancerous cell lines expressing variousendogenous p53 mutant proteins with TAR1 resulted in a significantincrease in apoptosis (FIGS. 17 a-r, Example 3). Moreover, cellsharboring the mutant p53 protein (R175H) which were stably transfectedto express the F2 scFv (TAR1) antibody of the present inventionexhibited reduced colony formation capability manifested in reducednumber of formed colonies as well as reduced size of the formed colonies(FIGS. 5, 6 a-d, Examples 3 and 4 of the Examples section whichfollows).

Thus, according to yet an additional aspect of the present invention,there is provided a method of inducing apoptosis and/or growth arrest ofcancer cells. The method is effected by contacting with or expressing inthe cancer cells the isolated polypeptide of the present invention(which is extensively described hereinabove), thereby inducing apoptosisand/or growth arrest of the cancer cells.

As used herein the term “apoptosis” refers to programmed cell deathwhereby the cell executes a “cell suicide” program. Apoptosis plays animportant role in a number of physiological events includingembryogenesis, regulation of the immune system, and homeostasis. Thus,apoptosis can be in response to diverse signals such as limb and neuraldevelopment, neurodegenerative diseases, radiotherapy and chemotherapy.Apoptotic processes are usually characterized by uncoupling ofmitochondrial oxidation, drop in levels of nicotinamide adeninedinucleotide phosphate [NAD(P)H], release of cytochrome c, activation ofcaspases, DNA fragmentation and externalization of phosphatidylserine (amembrane phospholipid normally restricted to the inner leaflet of thelipid bilayer) to the outer leaflet of the plasma membrane.

As used herein the phrase “growth arrest” refers to inhibition of cellgrowth in vitro, ex vivo (i.e., when cells are derived from anindividual and are cultured in a tissue culture) and/or in vivo (in atumor of an individual). In case in vitro or ex vivo conditions areused, the growth arrest can be detected by following colony formation(by counting the number of colonies), cell area, number of cells in acolony and the like using various methods known in the art. In case invivo conditions are utilized, the growth arrest can be also monitored byfollowing the size and shape of tumor and/or the presence of tumormetastases using various histological and immunological staining methodsknown to any one skilled in the art of pathology.

According to the method of this aspect of the present invention, thecancer cells in which apoptosis is induced according to the method ofthis aspect of the present invention specifically express the exposedepitope shared by mutant, but not wild type p53 protein (e.g., SEQ IDNO:1). Such cancer cells can harbor a severe or intermediate p53mutation as described hereinabove. Such cancer cells can be derived fromany kind of tumor, including solid tumors (e.g., breast cancer, coloncancer, lung cancer, hepatocellular carcinomas, osteogenic sarcomas,colorectal cancer, glioblastomas, esophageal carcinoma, bladder cancer,squamous cell carcinomas) and non-solid tumors (e.g., leukemia andlymphoma). In addition, such cells can be derived by mutagenesis ortransfection of normal cells with an oncogene using methods known in theart.

According to the method of this aspect of the present invention, theisolated polypeptide of the present invention is contacted with orexpressed in the cancer cells of the present invention. It will beappreciated that since p53 is expressed in the cell nuclei, the isolatedpolypeptide of the present invention is preferably a small polypeptide[e.g., a CDR-containing polypeptide or an scFv such as the F2 scFv (SEQID NO:113)] rather than a large molecule (such as an IgG molecule) whichcan therefore more easily penetrate the cell and nuclear membranes.

To facilitate the penetration of the isolated polypeptide to the cellnuclei, the isolated polypeptide can be covalently attached to ahydrophobic moiety or be expressed as a fusion gene with the hydrophobicmoiety (such as in the case of the TAT-scFv F2/H is expression plasmidsdescribed in the “General Materials and Experimental Methods” of theExamples section which follows). The hydrophobic moiety can be anysubstance which is nonpolar and generally immiscible with water such asa hydrophobic residue (portion) of a hydrophobic substance. Non-limitingexamples of hydrophobic substances include, but are not limited to,substituted and unsubstituted, saturated and unsaturated hydrocarbons,where the hydrocarbon can be an aliphatic, an alicyclic or an aromatic.Preferably, the hydrocarbon bears a functional group which enablesattachment thereof to an amino acid residue. Representative examples ofsuch functional groups include, without limitation, a free carboxylicacid (C(═O)OH), a free amino group (NH₂), an ester group (C(═O)OR, whereR is alkyl, cycloalkyl or aryl), an acyl halide group (C(═O)A, where Ais fluoride, chloride, bromide or iodide), a halide (fluoride, chloride,bromide or iodide), a hydroxyl group (OH), a thiol group (SH), a nitrilegroup (C≡N), a free C-carbamic group (NR″—C(═O)—OR′, where each of R′and R″ is independently hydrogen, alkyl, cycloalkyl or aryl), a freeN-carbamic group (OC(═O)—NR′—, where R′ is as defined above), a thionylgroup (S(═O)₂A, where A is halide as defined above) and the like. Forexample, such a hydrophobic moiety can be a fatty acid such as myristicacid, lauric acid, palmitic acid, stearic acid (C18), oleic acid,linolenic acid and arachidonic acid.

The isolated polypeptide of the present invention can be administered tothe cells using methods known in the art such as, the addition of theisolated polypeptide to the cell environment such as blood, plasma,buffers, tissue culture medium and the like.

Alternatively, the isolated polypeptide of the present invention can beexpressed using a nucleic acid construct in cells to form intrabodies asdescribed hereinabove.

Additionally or alternatively, the anti-p53 antibody of the presentinvention (e.g., the TAR1 antibody) can be presented on a viral displayvehicle such as a filamentous phage and be used in a therapeuticallyeffective amount to induce apoptosis and/or growth arrest of cancercells. The present inventors have previously demonstrated that suchviral display vehicles (i.e., phages) are inert vehicles and suitablefor carrying active antibody fragments to the CNS (U.S. Pat. Appl. No.20040013647 to Solomon, Beka et al.).

It will be appreciated that the viral display vehicle of the presentinvention can be designed to enable presentation of the p53 antibody ofthe present invention (e.g., TAR1) within a cell. As is shown in FIGS.18-21 and is described in Example 7 of the Examples section whichfollows, a viral display vehicle containing a PTD (protein transductiondomain) [e.g., the PEP peptide (SEQ ID NO:136) which enablesinternalization into a mammalian cell; shown in FIGS. 18 a-b] wascapable of penetrating into mammalian CHO cells. For penetration intothe nucleus, the viral vector can further include the NLS peptide (SEQID NO:134) conjugated to the displayed antibody. The constructed phagewill present a PTD on its coat protein VIII and the antibody fused tothe NLS peptide on its protein III (See FIGS. 18 a-b).

Thus, viral display vehicles which present the anti-p53 antibody of thepresent invention can be administered to an individual in need thereofand be targeted to the nuclei of the cells of interest. For example, forbrain tumors, the viral display vehicle can be administered to theindividual by intranasal application and thereby reach various brainregions such as the cortex and the hippocampus.

The apoptosis induced by the isolated polypeptide, the nucleic acidconstruct and/or the viral display vehicle of the present invention canbe detected using various methods known in the art.

Ethidium homodimer-1 staining—Apoptosis can be detected by dyesspecifically binding to cells with compromised membranes, i.e., deadcells. Briefly, unfixed cells such as cells in suspension, tissueculture, tissue sections and the like are stained with the fluorescentdye Ethidium homodimer-1 (excitation, 495 nm; emission, 635 nm). In thisassay, live cells have a green fluorescent cytoplasm but no EthD-1signal, whereas dead cells lack the green fluorescence and are stainedwith EthD-1.

Tunnel assay (Roche, Basel, Switzerland)—labels DNA breaks which arecharacteristics of cells undergoing apoptosis with fluorescein(excitation 450-500 nm, emission 515-565 nm).

Live/dead viability/cytotoxicity two-color fluorescence assay (MolecularProbes, Inc., L-3224, Eugene, Oreg., USA)—This assay measuresintracellular esterase activity with a cell-permeable substrate(Calcein-AM) which is converted by live cells to a fluorescentderivative (polyanion calcein, excitation, 495 nm; emission, 515 nm)which is thereafter retained by the intact plasma membrane of livecells.

FACS analysis—using molecules capable of specifically binding cellsundergoing apoptosis, such as propidium iodide and Annexin V. Annexins Vis a human protein characterized by calcium-mediated, high affinitybinding to phosphatidylserine which undergoes externalization to theouter side of the plasma membrane during early apoptosis.

DNA fragmentation by gel electrophoresis—Briefly, DNA is extracted fromunfixed cells and is subjected to gel electrophoresis (e.g., 1.5-2%agarose gel) and the degree of DNA fragmentation is evaluated using anyDNA stain such as Ethidium bromide, Syber Green and the like.

As is further shown in FIGS. 8 a-f and is described in Example 4 of theExamples section which follows, the F2 scFv (TAR1) antibody was capableof inhibiting tumor growth in vivo. These results strongly suggest theuse of the isolated polypeptide, the polynucleotide and/or expressionvector encoding same of the present invention in treating a p53-relatedcancer.

Thus, according to yet an additional aspect of the present inventionthere is provided a method of treating a subject suffering from or beingpredisposed to a p53-related cancer. The method is effected byadministering to or expressing in cells of the subject a therapeuticallyeffective amount of the isolated polypeptide of the present inventionthereby treating the p53-related cancer in the subject.

The term “treating” refers to inhibiting or arresting the development ofa disease, disorder or condition (e.g., a p53-related cancer) and/orcausing the reduction, remission, or regression of a disease, disorderor condition. Those of skill in the art will understand that variousmethodologies and assays can be used to assess the development of adisease, disorder or condition, and similarly, various methodologies andassays may be used to assess the reduction, remission or regression of adisease, disorder or condition.

As used herein, the term “subject” (or “individual” which isinterchangeably used herein) refer to a mammal, preferably a human beingat any age which suffers from a p53-related cancer. Preferably, thisterm encompasses individuals who are at risk to develop the p53-relatedcancer, i.e., individuals who are predisposed to a p53-related cancer.Such individuals can be carriers of a germ-line mutation in the p53 gene(GenBank Accession No. NM_(—)000546) such as in the case of Li-Fraumenisyndrome.

As used herein “p53-related cancer” refers to any cancer in which p53 isrelated to the onset or progression thereof. Such a cancer can be causedby a mutation in the p53 gene [GenBank Accession Nos. NC_(—)000017:7512464-7531642 (genomic region); NM_(—)000546 (mRNA); NP_(—)000537(protein)] leading to an abnormal structure and/or function of the p53protein. Such a mutation can be a missense, nonsense, splice mutation,promoter mutation, deletion, insertion, duplication and the like. As ismentioned hereinabove, various mutations in the p53 protein result inintermediate or severe conformational changes leading to abnormalfunction of the p53 protein. Non-limiting examples of p53-related cancerinclude those caused by germline mutations in the p53 gene (e.g., in thecase of Li-Fraumeni syndrome 1, OMIM #151623) as well as those caused bysomatic mutations in the p53 gene, such as hepatocellular carcinomas,osteogenic sarcomas, colorectal cancer, lung cancer, glioblastomas,esophageal carcinoma, bladder cancer, squamous cell carcinomas, leukemiaand lymphoma.

As used herein the phrase “cells of a subject” includes any cells whichare derived from the subject and are taken out of the subject (i.e., exvivo gene therapy) or cells which are part of the subject (i.e., in vivogene therapy as described hereinabove).

As used herein the phrase “therapeutically effective amount” refers toan amount of the isolated polypeptide of the present invention, thepolynucleotide or expression vector encoding the same and/or the viraldisplay vehicle which displays the anti-p53 antibody of the presentinvention, which is sufficient to exert the biological activity, i.e.,binding the exposed epitope of p53 (as described hereinabove), inducingapoptosis and treating or preventing the p53-related cancer.

As is shown in FIG. 5 and is described in Example 3 of the Examplessection which follows, the A4 scFv was capable of increasing thesusceptibility of cancer cells to chemotherapy drugs. These resultssuggest the use of the isolated polypeptide, isolated polynucleotide,the expression vector encoding same and/or a pharmaceutical compositionincluding same (as is further described hereinunder) together withconventional cancer treatment protocols utilizing e.g., chemotherapydrugs (i.e., combination therapy).

It will be appreciated that such a drug (e.g., a chemotherapy agent suchas Mechlorethamine, Fluorouracil, Dacarbazine, Docetaxel, Carmustine,Vindesine) can be also conjugated to the isolated polypeptide of thepresent invention or form a part of the expression vector encoding same.

In addition, to increase the specific biological activity exerted by theisolated polypeptide of the present invention such a polypeptide canfurther include a cytotoxic agent (i.e., a drug) such as Pseudomonasexotoxins PE35, PE38, PE40, Pseudomonas aeroginosa exotoxin A (ETA′),and diphtheria toxin (DT390), to thereby form a specific immunotoxin.Such a cytotoxic agent can be attached to the isolated polypeptide or bepart of polynucleotide expressed by the expression vector of the presentinvention.

Thus, according to one preferred embodiment of present invention, theisolated polypeptide of the present invention is fused or conjugated toa drug.

According to another preferred embodiment of the present invention theisolated polynucleotide and/or expression vector of the presentinvention further comprises an additional nucleic acid sequence encodinga drug.

It will be appreciated that such immunotoxins and/or chemotherapy agentscan be generated using recombinant DNA techniques (e.g., by ligating thecoding sequence of the agent molecule to the coding sequence of theisolated polypeptide of the present invention, usually downstream of thecoding sequence of the isolated polypeptide) or by covalentlyconjugating the toxin or chemotherapy agents to the isolated polypeptidesequence (e.g., to the polypeptide set forth by SEQ ID NO:113, 114, or115) by methods known in the art. For example by using a variety ofbifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bisazido compounds (such asbis-(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

The isolated polypeptide of the present invention, the polynucleotideand/or the nucleic acid construct encoding same can be administered toan organism per se, or in a pharmaceutical composition where it is mixedwith suitable carriers or excipients.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the active ingredients described herein with otherchemical components such as physiologically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to an organism.

Herein the term “active ingredient” refers to the recombinant isolatedpolypeptide of the present invention, the polynucleotide and/or theviral diaply vehicle accountable for the biological effect (i.e.,specific binding to the exposed epitope shared by mutant, but not wildtype p53 protein, inducing apoptosis and/or treating a p53-relatedcancer).

Hereinafter, the phrases “physiologically acceptable carrier” and“pharmaceutically acceptable carrier” which may be interchangeably usedrefer to a carrier or a diluent that does not cause significantirritation to an organism and does not abrogate the biological activityand properties of the administered compound. An adjuvant is includedunder these phrases.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of anactive ingredient. Examples, without limitation, of excipients includecalcium carbonate, calcium phosphate, various sugars and types ofstarch, cellulose derivatives, gelatin, vegetable oils and polyethyleneglycols.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, especially transnasal, intestinal or parenteraldelivery, including intramuscular, subcutaneous and intramedullaryinjections as well as intrathecal, direct intraventricular,intracardiac, intravenous, intraperitoneal, intranasal, or intraocularinjections.

Alternately, one may administer the pharmaceutical composition in alocal rather than systemic manner, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can beformulated readily by combining the active compounds withpharmaceutically acceptable carriers well known in the art. Suchcarriers enable the pharmaceutical composition to be formulated astablets, pills, dragees, capsules, liquids, gels, syrups, slurries,suspensions, and the like, for oral ingestion by a patient.Pharmacological preparations for oral use can be made using a solidexcipient, optionally grinding the resulting mixture, and processing themixture of granules, after adding suitable auxiliaries if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; cellulose preparations such as, for example, maize starch,wheat starch, rice starch, potato starch, gelatin, gum tragacanth,methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarbomethylcellulose; and/or physiologically acceptable polymers such aspolyvinylpyrrolidone (PVP). If desired, disintegrating agents may beadded, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acidor a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, titanium dioxide, lacquer solutions and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fitcapsules made of gelatin as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules may contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, lubricants such as talc ormagnesium stearate and, optionally, stabilizers. In soft capsules, theactive ingredients may be dissolved or suspended in suitable liquids,such as fatty oils, liquid paraffin, or liquid polyethylene glycols. Inaddition, stabilizers may be added. All formulations for oraladministration should be in dosages suitable for the chosen route ofadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for useaccording to the present invention are conveniently delivered in theform of an aerosol spray presentation from a pressurized pack or anebulizer with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichloro-tetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be determined by providing avalve to deliver a metered amount. Capsules and cartridges of, e.g.,gelatin for use in a dispenser may be formulated containing a powder mixof the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated forparenteral administration, e.g., by bolus injection or continuousinfusion. Formulations for injection may be presented in unit dosageform, e.g., in ampoules or in multidose containers with optionally, anadded preservative. The compositions may be suspensions, solutions oremulsions in oily or aqueous vehicles, and may contain formulatoryagents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active preparation in water-soluble form.Additionally, suspensions of the active ingredients may be prepared asappropriate oily or water based injection suspensions. Suitablelipophilic solvents or vehicles include fatty oils such as sesame oil,or synthetic fatty acids esters such as ethyl oleate, triglycerides orliposomes. Aqueous injection suspensions may contain substances, whichincrease the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may alsocontain suitable stabilizers or agents which increase the solubility ofthe active ingredients to allow for the preparation of highlyconcentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile, pyrogen-free waterbased solution, before use.

The pharmaceutical composition of the present invention may also beformulated in rectal compositions such as suppositories or retentionenemas, using, e.g., conventional suppository bases such as cocoa butteror other glycerides.

Pharmaceutical compositions suitable for use in context of the presentinvention include compositions wherein the active ingredients arecontained in an amount effective to achieve the intended purpose. Morespecifically, a therapeutically effective amount means an amount ofactive ingredients (the isolated polypeptide of the present invention orthe polynucleotide or expression vector encoding same) effective toprevent, alleviate or ameliorate symptoms of a disorder (the p53-relatedcancer) or prolong the survival of the subject being treated.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. For example, a dose can be formulatedin animal models to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals. The data obtained from thesein vitro and cell culture assays and animal studies can be used informulating a range of dosage for use in human. The dosage may varydepending upon the dosage form employed and the route of administrationutilized. The exact formulation, route of administration and dosage canbe chosen by the individual physician in view of the patient'scondition. (See e.g., Fingl, et al., 1975, in “The Pharmacological Basisof Therapeutics”, Ch. 1 p. 1).

Dosage amount and interval may be adjusted individually to provide thecancer cells expressing a mutant p53 protein [which results in exposureof the common epitope described hereinabove (e.g., SEQ ID NO:1)] withlevels of the active ingredient which are sufficient to exert thebiological activity (e.g., binding the exposed epitope of mutant p53described hereinabove, inducing apoptosis and/or treat a p-53-relatedcancer) (minimal effective concentration, MEC). The MEC will vary foreach preparation, but can be estimated from in vitro data. Dosagesnecessary to achieve the MEC will depend on individual characteristicsand route of administration. Detection assays can be used to determineplasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks oruntil cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the subject being treated, the severity of the affliction,the manner of administration, the judgment of the prescribing physician,etc.

Compositions of the present invention may, if desired, be presented in apack or dispenser device, such as an FDA approved kit, which may containone or more unit dosage forms containing the active ingredient. The packmay, for example, comprise metal or plastic foil, such as a blisterpack. The pack or dispenser device may be accompanied by instructionsfor administration. The pack or dispenser may also be accommodated by anotice associated with the container in a form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals, which notice is reflective of approval by the agency ofthe form of the compositions for human or veterinary administration.Such notice, for example, may be of labeling approved by the U.S. Foodand Drug Administration for prescription drugs or of an approved productinsert. Compositions comprising a preparation of the inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition, as is further detailed above.

It will be appreciated that introduction of nucleic acids of the presentinvention to the subject can be effected using any gene therapymethodology used in the art. Such as for example, by viral infectionwhich offers several advantages over other methods such as lipofection,since higher transfection efficiency can be obtained due to theinfectious nature of viruses.

Currently preferred in vivo nucleic acid transfer techniques (in vivogene therapy) include transfection with viral or non-viral constructs,such as adenovirus, lentivirus, Herpes simplex I virus, oradeno-associated virus (AAV) and lipid-based systems. Useful lipids forlipid-mediated transfer of the gene are, for example, DOTMA, DOPE, andDC-Chol [Tonkinson et al., Cancer Investigation, 14(1): 54-65 (1996)].The most preferred constructs for use in gene therapy are viruses, mostpreferably adenoviruses, AAV, lentiviruses, or retroviruses. A viralconstruct such as a retroviral construct includes at least onetranscriptional promoter/enhancer or locus-defining element(s), or otherelements that control gene expression by other means such as alternatesplicing, nuclear RNA export, or post-translational modification ofmessenger. Such vector constructs also include a packaging signal, longterminal repeats (LTRs) or portions thereof, and positive and negativestrand primer binding sites appropriate to the virus used, unless it isalready present in the viral construct. In addition, such a constructtypically includes a signal sequence for secretion of the peptide from ahost cell in which it is placed. Preferably the signal sequence for thispurpose is a mammalian signal sequence or the signal sequence of thepolypeptide variants of the present invention. Optionally, the constructmay also include a signal that directs polyadenylation, as well as oneor more restriction sites and a translation termination sequence. By wayof example, such constructs will typically include a 5′ LTR, a tRNAbinding site, a packaging signal, an origin of second-strand DNAsynthesis, and a 3′ LTR or a portion thereof. Other vectors can be usedthat are non-viral, such as cationic lipids, polylysine, and dendrimers.

It will be appreciated that since the isolated polypeptide of thepresent invention is capable of specifically binding the exposed epitopeshared by mutant, but not wild type, p53 protein, such a polypeptide canbe used in the diagnosis of a p53-related cancer in which such anepitope is exposed.

Thus, according to still an additional aspect of the present inventionthere is provided a method of diagnosing a p53-related cancer in asubject. The method is effected by: (a) contacting a biological sampleof the subject with the isolated polypeptide of the present inventionunder conditions suitable for immunocomplex formation between theisolated polypeptide and the exposed epitope shared by p53 mutantproteins and not by wild type p53 protein; and (b) detecting formationof the immunocomplex, thereby diagnosing the cancer in the subject.

As used herein the phrase “diagnosing” refers to classifying a disease(a p53-related cancer) or a symptom, determining a severity of thedisease, monitoring disease progression, forecasting an outcome of adisease and/or prospects of recovery.

As used herein “biological sample” refers to a sample of tissue or fluidisolated from a subject, including but not limited to, for example,blood cells, bone marrow cells and specifically macrophages, lymphfluid, various tumors, neuronal cells, dendritic cells, organs, and alsosamples of in vivo cell culture constituents. It should be noted that a“biological sample of the subject” may also optionally comprise a samplethat has not been physically removed from the subject. Preferably, thebiological sample used by the method of this aspect of the presentinvention is blood, lymph node biopsy, bone marrow aspirate and a tissuesample.

The cancer which is diagnosed using the method according to this aspectof the present invention can be any of the p53-related cancers describedhereinabove. Preferably, the biological sample obtained from the subjectis a blood sample, bone marrow aspirate, or a tissue specimen.

Diagnosis of cancer according to the present invention is effected bycontacting the biological sample of the subject with the isolatedpolypeptide of the present invention under conditions suitable forimmunocomplex formation.

As used herein the term “immunocomplex” refers to a complex formedbetween an antibody (e.g., the isolated polypeptide of the presentinvention) and its specific antigen (a p53 mutant protein in which thecommon epitope shared by p53 mutant proteins and not by wild type p53protein is exposed).

The immunocomplex of the present invention can be formed at a variety oftemperatures, salt concentration and pH values which may vary dependingon the isolated polypeptide used and the cancer cells and those ofskills in the art are capable of adjusting the conditions suitable forthe formation of each immunocomplex.

According to the method of this aspect of the present invention,detection of immunocomplex formation is indicative of a diagnosis of thep53-related cancer. Various methods can be used to detect theimmunocomplex of the present invention and those of skills in the artare capable of determining which method is suitable for eachimmunocomplex and/or the type of cells used for diagnosis.

For example, the immunocomplex can be detected by conventionalimmunohistochemistry or immunofluorescence, FACS, ELISA, Western blotand RIA analyses, or by a molecular weight-based approach.

Immunohistochemistry or immunofluorescence analyses—This method involvesdetection of an antigen (e.g., p53 mutant proteins harboring severe orintermediate mutations in which the common epitope shared by mutant, butnot wild type p53 is exposed) in situ in fixed cells by antigen specificantibody (i.e., the isolated polypeptide of the present invention). Theantigen specific antibodies may be enzyme linked or linked tofluorophores. Detection is by microscopy and subjective or automaticevaluation. If enzyme linked antibodies are employed, a colorimetricreaction may be required. It will be appreciated thatimmunohistochemistry is often followed by counterstaining of the cellnuclei using for example Hematoxyline or Giemsa stain.

Fluorescence activated cell sorting (FACS)—This method involvesdetection of an antigen in situ in cells by antigen specific antibodies.The antigen specific antibodies are linked to fluorophores. Detection isby means of a cell sorting machine which reads the wavelength of lightemitted from each cell as it passes through a light beam. This methodmay employ two or more antibodies simultaneously.

Enzyme linked immunosorbent assay (ELISA)—This method involves fixationof a sample (e.g., fixed cells or a proteinaceous solution) containingan antigen (e.g., p53 mutant proteins as described hereinabove) to asurface such as a well of a microtiter plate. An antigen specificantibody (i.e., the isolated polypeptide of the present invention)coupled to an enzyme is applied and allowed to bind to the antigen.Presence of the antibody is then detected and quantitated by acolorimetric reaction employing the enzyme coupled to the antibody.Enzymes commonly employed in this method include horseradish peroxidaseand alkaline phosphatase. If well calibrated and within the linear rangeof response, the amount of substrate present in the sample isproportional to the amount of color produced. A substrate standard isgenerally employed to improve quantitative accuracy.

Western blot—This method involves separation of a substrate (e.g., p53mutant proteins as described hereinabove) from other protein by means ofan acrylamide gel followed by transfer of the substrate to a membrane(e.g., nylon or PVDF). Presence of the substrate is then detected by anantibody specific to the substrate (i.e., the isolated polypeptide ofthe present invention), which are in turn detected by antibody bindingreagents. Antibody binding reagents may be, for example, protein A, orother antibodies such as those in the ECL kit (Amersham Biosciences Inc,Piscataway, N.J., USA). Antibody binding reagents may be radiolabeled orenzyme linked as described hereinabove. Detection may be byautoradiography, colorimetric reaction or chemiluminescence. This methodallows both quantitation of an amount of substrate and determination ofits identity by a relative position on the membrane which is indicativeof a migration distance in the acrylamide gel during electrophoresis. Itwill be appreciated that in the case of the MHC-peptide complex, anon-denaturing gel electrophoresis is preferably employed.

Radio-immunoassay (RIA): In one version, this method involvesprecipitation of the desired antigen (e.g., p53 mutant proteins asdescribed hereinabove) with a specific antibody (i.e., the isolatedpolypeptide of the present invention) and radiolabeled antibody bindingprotein (e.g., protein A labeled with I¹²⁵) immobilized on aprecipitable carrier such as agarose beads. The number of counts in theprecipitated pellet is proportional to the amount of antigen.

In an alternate version of the RIA, a labeled antigen and an unlabelledantibody binding protein are employed. A sample containing an unknownamount of antigen is added in varying amounts. The decrease inprecipitated counts from the labeled antigen is proportional to theamount of antigen in the added sample.

Molecular weight-based approach—It will be appreciated that theimmunocomplex formed between a p53 mutant protein as describedhereinabove and the isolated polypeptide of the present inventionexhibits a higher molecular weight than its components, i.e., theisolated polypeptide of the present invention or the a p53 mutantprotein. Thus, methods capable of detecting such a change in themolecular weight can be also employed. For example, the immunocomplexcan be detected by a gel retardation assay. Briefly, a non-denaturingacrylamide gel is loaded with samples containing the isolatedpolypeptide of the present invention and the p53 mutant protein beforeand after immunocomplex formation. A shift in the size (molecularweight) of the protein product as compared with its components isindicative of the presence of an immunocomplex. Such a shift to a highermolecular weight can be viewed using a non-specific protein stainingsuch as silver stain or Commassie blue stain. Alternatively, the p53mutant protein or isolated polypeptide of the present invention can belabeled (e.g., with a radioactive label) prior to gel electrophoresis.Additionally or alternatively, cells expressing the p53 mutant proteincan be radioactively labeled prior to protein extraction.

In order to facilitate detection of immunocomplex formation, thepolypeptide sequence of the isolated polypeptide of the presentinvention further comprises an amino acid sequence of a detectable label(i.e., an epitope tag). Such an amino acid sequence can be encoded by anucleic acid sequence which is included in the expression vector of thepresent invention.

According to additional preferred embodiments of this aspect of thepresent invention, the isolated polypeptide of the present invention isattached to a detectable label such as biotin, digoxigenin and the like.Such a detectable label can be covalently attached to the isolatedpolypeptide of the present invention using methods well known in thearts.

The agents described hereinabove for detection of immunocomplexformation may be included in a diagnostic kit/article of manufacturepreferably along with appropriate instructions for use and labelsindicating FDA approval for use in diagnosing and/or assessing aseverity of a p53-related cancer.

Such a kit can include, for example, at least one container including atleast one of the above described diagnostic agents (e.g., the F2, E6 orA4 scFv antibodies) and an imaging reagent packed in another container(e.g., enzymes, secondary antibodies, buffers, chromogenic substrates,fluorogenic material). The kit may also include appropriate buffers andpreservatives for improving the shelf-life of the kit.

Additional objects, advantages, and novel features of the presentinvention will become apparent to one ordinarily skilled in the art uponexamination of the following examples, which are not intended to belimiting. Additionally, each of the various embodiments and aspects ofthe present invention as delineated hereinabove and as claimed in theclaims section below finds experimental support in the followingexamples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., Ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(Eds.) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., Ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N.Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., Ed. (1994); Stites et al.(Eds.), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (Eds.), “Selected Methods inCellular Immunology”, W.H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., Ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,Eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., Eds. (1984); “Animal Cell Culture” Freshney, R. I., Ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

General Materials and Experimental Methods

Cells, Plasmids and Reagents—H1299 cells were obtained from AmericanType Culture Collection (Manassas, Va.) and were maintained in RPMI(Sigma, St. Louis, Mo.) supplemented with 10% fetal calf serum (Sigma).H1299-R175H cells stably expressing the hot spot mutation R175H wereobtained from V. Rotter (Weizmann Institute of Science). PlasmidspCMV/myc and pCMV/myc/nuc were provided by I. Benhar (Tel AvivUniversity). pCMV-scFv F2 and pCMV/nuc-scFv F2 were constructed byremoving the scFv F2 sequence from the corresponding pCC16 cloneisolated from the human synthetic library (Azriel-Rosenfeld et al 2004JMB 335, 177) and sub-cloning into the NcoI and Nod sites in pCMV andpCMV/nuc. The TAT-scFv F2/His expression plasmids were constructed bycloning the TAT peptide (RKKRRQRRRG; SEQ ID NO:133) into the NcoI siteupstream the scFv in the vector pCANTAB6-Fv (expressing a non-relevantscFv) or into the NdeI site upstream the MBP in the vectorpMalC-TNN-EGFP. Both vectors were provided by I. Benhar (Tel AvivUniversity). The Fv and the EGFP were replaced by scFv F2 cloned intothe NcoI and Nod sites of both plasmids. The TAT-scFv F2-NLS/Hisexpression plasmids were constructed by cloning the NLS peptide(DPKKKRKV; SEQ ID NO:134) into the Nod site downstream the scFv.

Library screening—To identify scFvs that specifically bind the mutantp53 common epitope, a human synthetic combinatorial library of arrayablesingle-chain antibodies was screened. The construction and properties ofthe library is described in Azriel-Rosenfeld et al 2004 JMB 335,177-192. The biopanning of the library was performed using biotinylatedFRHSVV (SEQ ID NO:1) and Dynabeads M280—streptavidin (Dynal).

Rescue of the library—An aliquot of the bacterial library glycerol stock(about 1×10¹⁰ clones) was inoculated into 2×YT containing 100 μg/mlampicillin and 1% glucose (YTAG), and Grown at 37° C. until the OD₆₀ nmwas 0.5. The cells were infected with M13KO7 helper phage at a ratio of1:20, incubated for 30 minutes at 37° C. without shaking and thentransferred to a shaking incubator 37° C. for additional 30 minutes. Theinfected cells were pelleted at 3,300 g for 10 minutes, resuspended in2×YT containing 100 μg/ml ampicillin and 50 mg/ml kanamycin (YTAK) andincubated overnight at 30° C. The cells were centrifuged at 4° C. for 10minutes at 8000 g. PEG/NaCl was added to the supernatant (in a 1:5 ratioof PEG/NaCl to supernatant) and was incubated for 1 hour on ice. Thephages were collected by a 30-minute centrifugation at 4° C. at 10,800 gand resuspended in PBS.

Biopanning—The streptavidin-dynabeads were equilibrated for 1 hour withMPBS (PBS containing 2% skim milk powder) on a rotator at roomtemperature. The beads were collected with a magnet and resuspended inMPBS containing 0.1% Tween-20. Phages were pre-incubated for 1 hour atroom temperature with streptavidin-dynabeads alone. These two stepsdeplete and avoid anti-streptavidin binders. The phages were transferredto a fresh tube and 5 nmol of biotinylated FRHSVV (SEQ ID NO:1) peptidewas added and incubated for 1 hour at room temperature on rotator. Theequilibrated dynabeads were added to the phage-antigen mixture andincubates for 15 minutes at room temperature on rotator. The tube wasleft in the magnetic rack for 1 minute and then carefully aspirated. Thebeads were washed 6 times with 1 ml MPBS containing 0.1% Tween-20. Thephages were then eluted from the beads using 1 ml 100 mM TEA(Triethylamine). The phages solution was immediately neutralized bytransferring to a tube containing 0.5 ml 1.0 M Tris (HCl) pH 7. Thepanning output was determined by plating serial dilutions of TG-1infected cells.

Monoclonal phage preparation—Individual colonies from the output plateswere picked and inoculated into 100 μl YTAG in sterile 96-well plates.The phages grew overnight at 30° C. with shaking at 150 rpm. A 10 μlinoculum was transferred to a second 96-well plate containing 200 μl ofYTAG per well and grown with shaking for 1 hour at 37° C. To each well25 μl YTAG containing 10⁹ plaque forming units (PFU) of helper phagewere added, incubated for 30 minutes at 37° C. without shaking, and foran additional 1 hour with shaking (150 rpm) at 37° C. The plates werespan for 10 minutes at 1,800 g, the medium was aspirated and the cellswere resuspended in 200 μl YTAK and grown overnight at 30° C. whileshaking (150 rpm). The plates were then span for 10 minutes at 1,800 gand 100 ml of the supernatant was used in phage ELISA.

ELISA—Plates were coated with 1 μg/well of BSA-Biotin and incubatedovernight at 4° C. Following each antibody incubation, the plates werewashed three times with PBST (PBS+0.05% Tween-20) followed by one washwith PBS. One μg/well of streptavidin was added, incubated for 1 hour at37° C. and washed as described above. The plate was divided in two, toone half the FRHSVV-Biotin (1 μg/well) was added, and to the second halfa BSA-Biotin (1 μg/well) was added (as a control). The plates wereincubated for 1 hour at 37° C., washed, blocked overnight at 4° C. with3% nonfat dry milk and scFv was added (the amount of scFvs variedbetween 100 ng to 500 ng for a 1 hour incubation at 37° C. Followingincubation with scFv, the plates were washed and the monoclonal anti M13(Amersham), monoclonal anti His-tag (Sigma) or monoclonal anti MBP(Sigma), depending on the source of the scFvs, were added (each a 1:1000dilution, according to manufacturer's instruction) for a 1-hourincubation at 37° C., followed by washes. Anti mouse IgG conjugated toHRP was applied, incubated for 1 hour at 37° C., washed and developedusing the substrate 3,3′,5,5′-tetramethylbenzidine (Sigma) according tomanufacturer's instructions. The plates were then scanned in theEasyReader 400 FW ELISA reader (SLT, Austria) at 450 nm.

Mutant and wild type core domain ELISA studies using the mAb 1620 orTAR1 antibodies—Plates were coated overnight at 4° C. with recombinantwild-type or mutant core domains or with PBS as control. The plates werewashed and blocked for 2 hours at 37° C. with 3% nonfat dry milk. mAb1620 or TAR1 antibodies were added and incubated for 1 hour at 37° C.followed by washes. Monoclonal anti MBP conjugated to HRP (dilution1:1000) was applied, incubated for 1 hour at 37° C., washed anddeveloped using the substrate 3,3′,5,5′-tetramethylbenzidine (Sigma)

Expression and purification of scFvs—scFv expression vectors wereprepared by fusing the TAT sequence (RKKRRQRRRG; SEQ ID NO:133) to theN-terminus of the scFv and three repeats of the NLS sequence (DPKKKRKV;SEQ ID NO:134) to the C-terminus of the scFv. E. coli cells, transformedwith pMalC-NN-TAT-scFv, pMalC-NN-TAT-scFv-NLS, pCANTAB6-TAT-scFv orpCANTAB6-TAT-scFv-NLS (all of the constructs are Histidine-tagged) weregrown in LB medium supplemented with 100 μg/ml ampicillin and 1% (w/v)glucose. When the culture reached A₆₀₀ of 0.6-0.9 it was induced with0.5 mM IPTG at 30° C. for four hour. Cell extracts were prepared in 20mM Phosphate buffer (pH 7.4), 0.5 M NaCl and 20 mM imidazole byfreezing-thawing followed by brief sonication. The extracts wereclarified by centrifugation at 20,000 g and scFv was purified on aHisTrap HP column (Amersham Biosciences) using an AKTA prime (Amershampharmacia biotech) according to the manufacturer's instructions.

BIAcore Analysis—Real time analysis of the interaction between scFvs andFHRSVV-biotin were determined using BIAcore technology (Biacore AB,Uppsala, Sweden) according to manufacturer's instructions. The peptideFRHSVV-biotin was immobilized on a streptavidin coated sensor chipsurface SA. The amount of immobilized peptide was 200 pg/mm², thebinding was in HBS buffer (10 mM Hepes PH 7.4, 150 mM NaCl, 0.005%Tween-20, 3.4 mM EDTA). Several concentrations of purified scFv (62 nM,125 nM, 250 nM, 1 μM and 2 μM) were injected at a flow rate of 20μl/minute. Dissociation was observed for 180 seconds in running buffer(HBS). Regeneration of the sensor chip was performed using 10 μl of 10mM HCl. The kinetics parameters of the binding reactions were determinedby BIAevaluation 4.0 Software (Biacore AB) using the 1:1 Langmuir model.The dissociation rate (off-rate) constant Kd and the association rateconstant (on-rate) Ka were determined simultaneously according tomanufacturer's instructions.

FACS analysis—Each well was seeded with 3×10⁵ Cells. Twenty-four hourslater purified TAT-scFV F2-NLS (250 nM, 0.5 μM, 1 μM or 2 μM) was addedto the medium and incubated for additional 24 hour. Cells were harvestedby combining the detached cells in the supernatant with the adherentcells from the same well that had been removed by trypsinization. Thecells were pelleted, washed with PBS and then fixed by slowly adding 1ml of cold 70% ethanol. The cells were kept overnight at 4° C., pelletedonce again, resuspended in 1 ml PBS and stained by adding 50 μl ofpropidium iodide (1 mg/ml). The intensity of staining was determinedusing a FACScalibur Flow Cytometry System (Becton Dickinson).

Establishment of scFv expressing cells—H1299-R175H cells weretransfected with pCMV/myc-scFv-F2 plasmid using Fugene (Roche) at aratio of 2 μg DNA:4 μl Fugene, according to the manufacturer'sinstructions. Forty-eight hours post transfection the selectable drugG418 (Invitrogen) was added at a concentration of 0.4 mg/ml. The cellswere grown in the presence of G418 for 3 weeks to achieve colonies ofsingle cell clones. Each colony was expended and tested for scFv F2expression by western blot analysis.

Western Blot Analysis—Cells were lysed in a passive lysis buffer(Promega). Protein concentration was determined using the BCA ProteinAssay Kit (Pierce). Lysate aliquots were resolved by SDS-PAGE on 10%polyacrylamide gel, transferred to a nitrocellulose membrane and probedwith anti MBP-HRP (Sigma) or anti His-HRP (Sigma). Membranes were thendeveloped using the ECL kit (Amersham Biosciences, Uppsala, Sweden).

Immunoprecipitation—For immunoprecipitation studies of the mutant orwild type core domains, the proteins were incubated over night with 80nM TAR1. Immunoprecipitation was performed with mAb DO-12 antibody(dilution 1:33) and protein G magnetic beads incubated over night at 4°C. Beads were washed with PBS, sample buffer was added and boiled for 5minutes. The eluted proteins were subjected to Western blot analysisusing anti His-HRP (dilution 1:3000) and developed using the ECL kit.

Colony formation—H1299-R175H stably expressing mutant p53 orH1299-R175H-scFv F2 stably expressing both mutant p53 and scFv F2 wereseeded at low concentration (500-1000 cells/plate). The cells were grownfor two weeks, the medium was aspirated and colonies were stained withGiemsa. The number and size of colonies was determined using the analyzeparticle tool of the scion (NIH Image Beta 4.0.2) analysis system.

Animal studies—For the assessment of the anti-tumor activity of scFv F2,nine CD1 nude mice (six weeks old) were inoculated with 5×10⁶H1299-R175H cells subcutaneously and unilaterally into the right flanks.After 3 days mice were divided into two groups. One group of 5 micereceived intratumor injections of scFv F2 at a dose of 200 μg/mouse, andthe second group of 4 mice were injected with PBS and served as control.Injections were given every other day for two weeks. The tumor size wasmeasured and the tumor volume was calculated using the formula (a²×b)/2,where (a) is the short axis and (b) is the long axis. Relative volumefor each group was determined by dividing the average tumor volume foreach data point by the average starting tumor volume.

Circular dichroism measurements—A tandem cuvette (Hellma) was usedallowing simultaneous measurement of both p53 core domains and TAR1separately or in complex. All proteins were diluted to a concentrationof 1 μM in 50 mM Tris, 150 mM NaCl. Circular dichroism absorbancemeasurements were taken in an AVIV instrument. The experiment parameterswere as follows: Spectrum scan of 190 nm-250 nm; 1 nm step; 5 secondsmeasurement average in each step; 3 scans were taken for each sample.Measurements were taken at 4° C.

TUNEL assay—H1299 cells null for p53 or H1299 cells expressing R175Hmutant p53 were grown on slides. Cells were incubated overnight in theabsence or presence of 1 μM TAR1. Slides were washed with PBS and fixedfor 10 minutes at room temperature with 3.7% formaldehyde followed bywash with PBS. Samples were permeabilized with Cytonin for 15 minutes atroom temperature and than washed with water. Quenching of endogenousperoxidase was done with 3% H₂O₂ in methanol for 5 minutes at roomtemperature. Slides were washed with PBS and TdT labeled for 1 hour at37° C. with biotinylated nucleotides followed by wash with PBS. Slideswere incubated for 10 minutes with Streptavidin-HRP washed twice withPBS and developed with the substrate diaminobenzidine.

Example 1 SCFVs Bind the Common Epitope of Mutant P53 with High Affinity

To identify scFvs that specifically bind the mutant p53 common epitope,a human synthetic combinatorial library of arrayable single-chainantibodies (Azriel-Rosenfeld et al 2004 MB 335, 177-192) was screenedusing the biotinylated FRHSVV (SEQ ID NO:1) peptide and the streptavidinDynabeads (Dynal).

Twenty scFv clones were selected by biopanning using the common epitopeof mutant p53—FIGS. 1 a-b depict the amino acids sequences of thevariable Heavy chain (V_(H)) and variable light chain (V_(L)) of theisolated scFvs selected by biopanning with the FRHSVV (SEQ ID NO:1)peptide of the common epitope of the mutant p53. Table 3, hereinbelow,depicts the amino acid sequences of the CDR1, CDR2 and CDR3 of thevariable heavy chain of all scFv clones isolated and described in FIGS.1 a-b and Tables 4 and 5.

TABLE 3 Variable heavy chain CDR1 CDR2 CDR3 FSGYWMHWV EISGSGDSTHYGDSVKGGRNGSLDYW (SEQ ID NO: 5) (SEQ ID NO: 6) (SEQ ID NO: 7) Table 3: Theamino acid sequence of the CDR1, CDR2 and CDR3 of the variable heavy(VH) chain of all the isolated scFv clones of the present invention (forscFv clone IDs see Table 2, hereinbelow).

Tables 4 and 5, hereinbelow, present the CDRs of the variable lightchain of the isolated scFvs (Table 4) and the unique CDRs of thevariable light chains (Table 5) of the isolated scFvs selected againstthe FRHSVV (SEQ ID NO:1) peptide.

TABLE 4 variable light chain scFv Clone ID CDR1 CDR2 CDR3 No.TGSSSNIGADYDVHW IYDNHKRPSGV CQSWDNSAVVFGGGTQL G4 SEQ ID NO: 8)(SEQ ID NO: 9) (SEQ ID NO: 10) TGSSSNIGAGYDVHW IYSNHHRPSGVCQVWDSSSDHVVFGGGTQL G11 (SEQ ID NO: 11) (SEQ ID NO: 12) (SEQ ID NO: 13)TGSSSNIGAGYDVHW IYDNSHRPSGV CQVWDSSSEHVEFGGGTQL H8 (SEQ ID NO: 14)(SEQ ID NO: 15) (SEQ ID NO: 16) TGTTSNIGAGYDVHW IYKNDKKKSGVCASWDDSLNGHVVFGGGTQL B11 (SEQ ID NO: 17) (SEQ ID NO: l8) (SEQ ID N0: 19)TGSSSNIGADYDVHW IYGNYHRPSGV CSSWDDSQSGHVAFGGGTQV A2 (SEQ ID NO: 20(SEQ ID NO: 21) (SEQ ID NO: 22) TGSSSNIGADYDVHW IYDNDKRPSGVCAAWDDSLSGPVFGGGTQV A3 (SEQ ID NO: 111) (SEQ ID NO: 112) (SEQ ID NO: 23)TGSSSNIGADYDVHW IYDNDKRPSGV CAVWDDSLNAVVFGGGTKV C6 (SEQ ID NO: 24)(SEQ ID NO: 25) (SEQ ID NO: 26) TGSSANIGAGYDVHW IYDNDKRPSGVCAAWDDRLSGVVFGGGTKV B6 (SEQ ID NO: 27) (SEQ ID NO: 28) (SEQ ID NO: 29)TGNSSNIGAGYDVHW IYSNNQRPSGV CAAWDDSLNGPVPGGGTQV A5 (SEQ ID NO: 30)(SEQ ID NO: 31) (SEQ ID NO: 32) TGSSSNIGAGYDVHW IYANNNRPSGVCAAWDDNLNGLVFGGGTQL D12 (SEQ ID NO: 33) (SEQ ID NO: 34) (SEQ ID NO: 35)TGNSSNIGAGYDVHW IYSNNQRPSGV CAAWDDSLSSVVFGGGTKL A4 (SEQ ID NO: 36)(SEQ ID NO:37) (SEQ ID NO: 38) SGTSSNIGADYDVHW IYDNNKRPSGVCAAWDSSLSSVVFGGGTQL F2 (SEQ ID NO: 39) (SEQ ID NO: 40) (SEQ ID NO: 4l)SCSSSNIGADYDVHW IYSNNQRPSGV CAAWDDSLNGYVFGTGTKL E4 (SEQ ID NO: 42)(SEQ ID NO: 43) (SEQ ID NO: 44) SGSSSNIGAGYDVHW IYGNTNRPSGVCQSFDSTLSGPVFGGGTKV E6 (SEQ ID NO: 45) (SEQ ID NO: 46) (SEQ ID NO: 47)SGSSSNIGAGYDVHW IYGNTNRPSGV CAAWDDSLNGLVFGGGTQV F6 (SEQ ID NO: 48)(SEQ ID NO: 49) (SEQ ID NO: 50) SGSNSNIGAGYDVQW IYANSNRPSGVCGVWDDSLNGPVFGGGTPV D10 (SEQ ID NO: 51) (SEQ ID NO: 52) (SEQ ID NO: 53)TGGSSNIGNHHVSW IYGNTNRPSGV CQVWDSSSDHVVFGGGTKV D4 (SEQ ID NO: 54)(SEQ ID NO: 55) (SEQ ID NO: 56) SGTSSNIGSHTVHW IYEVNKRPSGVCQSYDSSLSAVVFGGGTQV F7 (SEQ ID NO: 57) (SEQ ID NO: 58) (SEQ ID NO: 59)TGGRFNIGDYAVHW IYDNDRRPSGV CAAWDDSLDGLVFGGGTQL A6 (SEQ ID NO: 60)(SEQ ID NO: 61) (SEQ ID NO: 62) TGGRFNIGDYAVHW IYDNDRRPSGVCQSFDSTLSGPVFGGGTKV C5 (SEQ ID NO: 63) (SEQ ID NO: 64) (SEQ ID NO: 65)Table 4: The amino acid sequence of the CDR1, CDR2 and CDR3 of thevariable light chain of the various scFv clones are presented (SEQ IDNOs: 8-65 and 111-112).

TABLE 5 Light chain Unique CDRs CDR1 CDR2 CDR3 TGSSSNIGADYDVHWIYDNHKRPSGV SWDNSAVVFGGGTQL (SEQ ID NO: 66) (SEQ ID NO: 67)(SEQ ID NO: 68) TGSSSNIGAGYDVHW IYSNHHRPSGV QVWDSSSDHVVFGGGTQL(SEQ ID NO: 69) (SEQ ID NO: 70) (SEQ ID NO: 71) TGTTSNIGAGYDVHWIYDNSHRPSGV QVWDSSSEHVEFGGGTQL (SEQ ID NO: 72) (SEQ ID NO: 73)(SEQ ID NO: 74) TGSSANIGAGYDVHW YENDKRPSGV ASWDDSLNGHVVFGGGTQL(SEQ ID NO: 75) (SEQ ID NO: 76) (SEQ ID NO: 77) TGNSSNIGAGYDVHWYGNYHRPSGV SSWDDSQSGHVAFGGGTQV (SEQ ID NO: 78) (SEQ ID NO: 79)(SEQ ID NO: 80) SGTSSNIGADYDVHW YDNDKRPSGV AAWDDSLSGPVFGGGTQV(SEQ ID NO: 81) (SEQ ID NO: 82) (SEQ ID NO: 83) SGSSSNIGADYDVHWYSNNQRPSGV AVWDDSLNAVVFGGGTKV (SEQ ID NO: 84) (SEQ ID NO: 85)(SEQ ID NO: 86) SGSSSNIGAGYDVHW ANNNRPSGV AAWDDRLSGVVFGGGTKV(SEQ ID NO: 87) (SEQ ID NO: 88) (SEQ ID NO: 89) SGSNSNIGAGYDVQWYDNNKRPSGV AAWDDSLNGPVFGGGTQV (SEQ ID NO: 90) (SEQ ID NO: 91)(SEQ ID NO: 92) TGGSSNIGNHHVSW GNTNRPSGV AAWDDNLNGLVFGGGTQL(SEQ ID NO: 93) (SEQ ID NO: 94) (SEQ ID NO: 95) SGTSSNIGSHTVHW ANSNRPSGVAWDDSLSSVVFGGGTKL (SEQ ID NO: 96) (SEQ ID NO: 97) (SEQ ID NO: 98)TGGRFNIGDYAVHW EVNKRPSGV AAWDSSLSSVVFGGGTQL (SEQ ID NO: 99)(SEQ ID NO: 100) (SEQ ID NO: 101) IYDNDRRPSGV AAWDDSLNGYVFGTGTKL(SEQ ID NO: 102) (SEQ ID NO: 103) CQSFDSTLSGPVFGGGTKV (SEQ ID NO: 104)CAAWDDSLNGLVFGGGTQV (SEQ ID NO: 105) NGPVFGGGTPV (SEQ ID NO: 106)CQVWDSSSDHVVFGGGTKV (SEQ ID NO: 107) LSAVVFGGGTQV (SEQ ID NO: 108)CAAWDDSLDGLVFGGGTQL (SEQ ID NO: 109) CQSFDSTLSGPVFGGGTKV(SEQ ID NO: 110) Table 5: The unique amino acid sequences of the CDR1,CDR2 and CDR3 of the variable light chain of the various scFv clones arepresented (SEQ ID NOs: 66-110).

ScFvs F2, A4 and B6 are specific to the common epitope of mutant p53—TheF2, A4 and B6 scFvs were tested for their binding capacity to the BovineSerum Albumin (BSA) and Streptavidin proteins and the FRHSVV (SEQ IDNO:1), 13 amyloid peptide DAEFRHDSGYEVHHQK SEQ ID NO:2), MAP-PrP(DYEDRYYRE; SEQ ID NO:3) and the PaP (ILLWQPIPV; SEQ ID NO:4) peptides.As is shown in FIG. 9, the F2 scFv, A4 scFv and to a lesser extent, theB6 scFv exhibited high affinity towards the peptide derived from thecommon epitope of mutant p53. On the other hand, these antibodiesexhibited low affinity towards the other peptides or proteins tested.

The scFv F2 and E6 antibodies display high affinity binding of themutant epitope—To determine the affinity binding of the isolated scFvstowards the target peptide (SEQ ID NO:1), the BIAcore technology wasemployed. A biotin conjugate of the peptide (FRHSVV; SEQ ID NO:1) wasimmobilized on a streptavidin coated sensor chip surface and the bindingspecificity was determined using increasing concentrations of thepurified F2 and E6 scFvs. As is shown in FIGS. 2 a-b, both the F2 (FIGS.2 a) and E6 (FIG. 2 b) scFvs were capable of binding the common epitopeof the mutant p53 with an affinity of 1.1×10⁻⁸ and 4.6×10⁻¹⁴ M,respectively. These results demonstrate, for the first time, theisolation of human antibodies directed against the common epitope of p53with an affinity suitable for therapeutic applications.

The F2 and A4 antibodies are specific to the common epitope of mutantp53—As is shown in F1G. 9, ELISA performed using the F2, A4 and B6 scFvsrevealed that the F2 and A4 antibodies are highly specific to thepeptide derived from the common epitope of mutant p53 (SEQ ID NO:1).

Example 2 The Isolated SCFVs Bind Whole Mutant P53 Proteins with SevereConformational Changes

To demonstrate the binding capacity of the isolated scFvs to whole p53molecules, ELISA plates were coated with recombinant wild-type p53 ormutant p53-R175H, p53-R248H and p53-R273W whole proteins which wereproduced in sf9 insect cells and 100-500 ng scFv F2 were added.

As is shown in FIG. 3, the F2 scFv (also designated herein as TAR1)purified antibody differentially bound to the various p53 proteins.While binding of the F2 scFv to the wild type p53 protein was relativelylow (0D₄₉₂ nm ˜0.1), binding to the p53 mutants exhibiting a severeconformational change (e.g., R175H) or an intermediate conformationalchange (e.g., R248W) was significantly high (0D₄₉₂ nm>0.25). Thus, thesevere conformational change in p53 protein which results in a higherexposure of the common mutant epitope is more susceptible to theantibodies of the present invention.

These results suggest the use of the antibody of the present invention(e.g., F2 scFv) to specifically and differentially target mutant p53proteins without affecting wild-type p53 proteins.

Example 3 The F2 scFv Antibody (TAR1) Induces Apoptosis and InhibitColony Formation in Cells Expressing Mutant P53

The apoptotic activity of wild type p53 is a major contributor to itstumor suppressor function. However, this activity is lost when p53 ismutated. To determine whether F2 SCFV (TAR1) can restore the apoptoticfunction to mutant p53, cancer cell lines stably expressing the mutantR175H p53 protein were treated with TAR1 and the effect on apoptosis wasdetermined using the FACS and TUNEL analyses, as follows.

F2 scFv (TAR1) is capable of inducing apoptosis—The purified isolated F2scFv antibody (TAR1) was tested for its capacity to induce apoptosis inH1299 human lung carcinoma cells expressing the mutant R175H p53protein. As is shown in FIGS. 4 a-j and 15 a-d, while in cellsexpressing the mutant R175H p53 protein treatment for 24 hours with theF2 scFv antibody (TAR1) caused apoptosis in a dose-dependent manner(e.g., ˜13% and ˜32% of apoptotic cells in the presence of 0.5 and 2 μMF2 scFv, respectively; FIGS. 4 a-e and FIG. 15 a-b), in lung carcinomacells not expressing the R175H p53 protein, the F2 scFv antibody failedto induce apoptosis (FIGS. 4 f-j and FIGS. 15 c-d).

Similar results were obtained when HCT116 colon carcinoma cells stablyexpressing mutant p53 R175H, were treated for 24 hours with 1 μM TAR1.As is shown in FIGS. 15 e-h, TAR1 caused a substantial increase in thefraction of cells with a sub-G1 DNA content, indicating DNAfragmentation and cell death (FIG. 15 f). Induction of apoptosis was notapparent in HCT116 cells expressing wild-type p53 (FIG. 15 h) indicatingthat induction of apoptosis by TAR1 is mutant p53 dependent.

These results were corroborated by the TUNEL assay shown in FIGS. 16a-d. Staining of apoptotic cells was evident only in H1299 cellsexpressing mutant p53 R175H that were treated with TAR1 (FIG. 16 b) butnot in untreated cells (FIG. 16 a). No staining was observed in H1299cells null for p53 regardless whether cells were treated with TAR1 (FIG.16 d) or not (FIG. 16 c).

TAR1 is capable of inducing cell death by apoptosis in cancerous celllines expressing endogenous mutant p53 proteins—The effect of TAR1 oncell death was further tested in a panel of nine human tumor cell linesendogenously expressing different point mutations in their p53 coredomains, representing various tumor types including colon (KM12-C,SW480, Colon 320), breast (T47D, SKBR3, MCF7, MDA231), brain (e.g.,neuroblastoma LAN1) and pancreas (PANC1). Cells were treated for 24hours with 1 μM TAR1 (FIGS. 17 b, d, f, h, j, l, n, p, r) or remaineduntreated (FIGS. 17 a, c, e, g, i, k, m, o, q) and the effect onapoptosis was assessed using FACS analysis. As is shown in FIGS. 17 a-r,TAR1 treatment resulted in a differential induction of apoptosis in alltested cell lines regardless of the nature of their point mutation orthe tumor type, as manifested by the accumulation of cells in the sub-G1fraction.

Cells expressing the F2 scFv exhibit reduced colony formation—To furthersubstantiate the effect of the F2 scFv antibody on apoptosis and cellgrowth, cells harboring the mutant p53 protein (R175H) were stablytransfected with an expression vector harboring the F2 scFv antibody andthe effect on cell growth was determined in tissue cultures seeded with500-1000 cells/plate. As is shown in FIGS. 6 a-d, both the number ofcolonies and colony area were significantly reduced in cells expressingthe F2 scFv antibody.

Altogether, these results demonstrate that the F2 scFv antibody of thepresent invention can induce apoptosis and inhibit colony formation.These results suggest the use of the scFv antibodies of the presentinvention in treating cancer in cells expressing mutant p53.

The F2 scFv increases susceptibility of cells expressing mutant p53 tochemotherapy drugs—To test the potential use of the scFv antibodies ofthe present invention in combination with common chemotherapy drugs, theeffect of the etoposide and cisplatin drugs was tested on cellsexpressing mutant p53 in the presence or absence of the F2 scFvantibody. As is shown in FIG. 5, while cells expressing the F2 scFvantibody were highly susceptible to the chemotherapy drugs atconcentrations as low as 0.5 μg/ml of cisplatin or 1 μM of etoposide,human lung carcinoma cells not expressing the scFv antibody were lesssusceptible towards such drugs.

These results further suggest the use of the scFv antibodies of thepresent invention in treating cancer in combination with other drugs.

Example 4 The F2 scFv Antibody Suppresses Tumor Growth In vivo

To test the ability of the F2 scFv antibody of the present invention toinhibit tumor cell growth in vivo, nude mice were subject tosubcutaneous injection of human lung carcinoma cell (H1299-R175H). Threedays later, the mice received an intra-tumor injection of scFv F2 (200μg) or PBS (50 μl) which was repeated every other day for two weeks. Asis shown in FIG. 7, while in the PBS-treated mice the tumor sizedrastically increased within 28-36 days post inoculation, in the F2scFv-treated mice, the tumor size remained unchanged, even following 36days. FIGS. 8 a-f depict the suppression of tumors in F2 scFv-treatedmice. Altogether, these results demonstrate the use of the purified F2scFv antibody in suppressing tumor growth in vivo.

Example 5 Restoration of Wild-Type P53 Conformation to Mutant P53

The present inventors employed biochemical and biophysical methods todetermine whether binding of TAR1 (i.e., the F2 scFV antibody) to mutantp53 can restore the wild-type protein folding, as follows.

Experimental Results

TAR1 increases the binding of mAb 1620, a wild type p53-specificantibody, to the p53 R175H mutant—Recombinant polypeptides of the P53core domain (including amino acids 94-312 of the p53 protein set forthby SEQ ID NO:135; GenBank Accession No. NP_(—)000537) of wild-type andR175H mutant P53 proteins were subjected to ELISA andimmunoprecipitation assays using conformation specific monoclonalantibodies. The first step was to establish that the purifiedrecombinant core domain proteins exhibit the specific conformation ofwild-type and mutant p53 proteins. An ELISA assay using TAR1 antibody(which recognizes the same epitope as mAb 240), which is specific to themutant conformation (FIG. 10 a) and the mAb 1620 (Abcam Laboratories,Ltd, UK), which is specific to the wild-type conformation (FIG. 10 b),confirmed that this indeed was the case. Binding of TAR1 to mutant p53R175H and wild-type core domains upon heating for 30 or 60 minutes at37° C. resulted in a three-fold increase in the mAb 1620 positivefraction of the mutant core domain and prevention of unfolding of thewild-type core domain and stabilization of wild-type p53 conformation(FIGS. 10 c and d). To substantiate these results the mAb DO-12(Novocastra Laboratories, Ltd, UK), which is specific for the mutant p53conformation, yet binds to a different epitope than TAR1, was employedin an immunoprecipitation assay. Binding of TAR1 to mutant p53 coredomain caused a dramatic decrease in the DO-12 positive fraction, buthad no effect on wild-type core domain (FIG. 11 a).

Binding with TAR1 causes a shift in the spectrum of mutant p53—Circulardichroism is a method for analyzing protein secondary structure insolution and can determine whether protein-protein interaction alter theconformation of the protein. Conformational changes in the proteinsresult in a spectrum that is different from the sum of the individualcomponents. The circular dichroism method was employed using the TAR1antibody and wild-type p53 or mutant R175H core domains. Binding of TAR1caused a shift in the spectrum of mutant p53 while almost no differencewas observed in the spectrum of the wild-type core domain indicating aconformational change in the complex of TAR1 and mutant p53. The spectraof both TAR1 complexes, with wild-type p53 and with mutant p53, are verysimilar indicating conformational similarity (FIG. 12).

TAR1 treatment of cells expressing mutant p53 increases the binding ofmAb 1620 to extract of such cells—As is shown in FIG. 11 b, a change inconformation of mutant p53 could be detected in H1299 cells stablyexpressing mutant p53 R175H. Upon treatment of these cells with TAR1 anincrease in the fraction of cells adopting wild-type conformation wasobserved, as judged by immunoprecipitation with the wild-type specificmAb 1620.

Altogether, these results demonstrate that TAR1 is capable of restoringwild type conformation to mutant p53 core domain.

Example 6 TAR1Restores Transcriptional Transactivation to Mutant P53

Loss of the ability to transactivate transcription of wild-type p53target genes is one of mutant p53 characteristic features. The presentinventors have tested the ability of TAR1 to restore P53 transcriptionaltransactivation function in cells expressing mutant p53, as follows.H1299 cells stably expressing mutant p53 R175H protein were treated for24 hours with TAR1 and the expression level of p21, MDM2 and Bax wasexamined by Western blot analyses. Treatment of cells with TAR1 resultedin an increase in the expression level of three endogenous wild-type p53target genes p21 (FIG. 13 a), MDM2 (FIG. 13 b) and Bax (FIG. 13 d) in aconcentration dependent manner. The expression level of tubulin servedas an internal control for protein levels used in each experiment (FIGS.13 e-h). Furthermore, some mutant p53 proteins have been shown totransactivate transcription of genes different from those activated bywild-type p53, one of them is EGR1. As is shown in FIG. 13 c, treatmentof the cells with TAR1 abrogated the gain of function activity of themutant p53 protein as manifested by the lower expression of EGR1 in thetreated cells.

Thus, these results demonstrate, that the TAR1 antibody of the presentinvention is capable of restoring the transcriptional transactivationfunction of wild-type specific target genes (e.g., p21, MDM2 and Bax)and preventing the transcriptional activation of mutant P53.

Example 7 Viral Display Vehicles are Useful Therapeutic Agents

The present inventors have designed a viral display vehicle capable ofpenetrating into mammalian cells (e.g., CHO cells) which can be used todeliver the anti-p53 antibody of the present invention (e.g., TAR1) intocell-of-interest, as follows.

A filamentus phage, fuse 88, expressing a PTD (e.g the peptide PEP asshown in FIGS. 18 a-b) on the coat protein VIII. The PEP peptide (SEQ IDNO:136) enables the penetration of the phage into the cell. Thefilamentus phage also includes the scFv of the present invention (e.g.,TAR1) fused to an NLS (SEQ ID NO:134) on protein III. The NLS peptideenables nuclear localization of the phage. The results presented inFIGS. 18-21 demonstrate that the phage PEP is capable of penetratingmammalian cells and has no toxic effect on these cells. Such afilamentus phage (i.e., the viral display vehicle) can be administeredintranasally as a therapeutic agent for treatment of brain tumors.

Thus, these results demonstrate the use of the viral display vehiclewhich includes the anti-p53 antibody of the present invention for thetreatment of p53 related diseases.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims. All publications, patents and patentapplications mentioned in this specification are herein incorporated intheir entirety by reference into the specification, to the same extentas if each individual publication, patent or patent application wasspecifically and individually indicated to be incorporated herein byreference. In addition, citation or identification of any reference inthis application shall not be construed as an admission that suchreference is available as prior art to the present invention.

REFERENCES Additional References are Cited in Text

-   1. Govorko D, Cohen G and Solomon B., 2001, J. Immunol. Methods.    258: 169-81.-   2. Hupp T. R and Lane D. P., 1994, Curr. Biol. 4: 865-875.-   3. Gannon J V, et al., 1990, EMBO J. 9(5): 1595-602.-   4. Govorko D, Cohen G and Solomon B., 2001, J. Immunol. Methods.    258: 169-81.-   5. Azriel-Rosenfeld R, et al., 2004, J. Mol. Biol. 335(1): 177-92.

1. An isolated polynucleotide comprising a nucleic acid sequenceencoding an antibody polypeptide that specifically binds an exposedepitope shared by p53 mutant proteins and not by wild type p53 protein,wherein said polypeptide has affinity for said epitope of less than 25nanomolar, and wherein said polypeptide comprises the amino acidsequences set forth in SEQ ID NO:5 (CDR1), SEQ ID NO:6 (CDR2) and SEQ IDNO:7 (CDR3) in a heavy chain of said antibody and the amino acidsequences set forth in SEQ ID NO:45 (CDR1), SEQ ID NO:46 (CDR2) and SEQID NO:47 (CDR3) in a light chain of said antibody; or the amino acidsequences set forth in SEQ ID NO:5 (CDR1), SEQ ID NO:6 (CDR2) and SEQ IDNO:7 (CDR3) in a heavy chain of said antibody and the amino acidsequences set forth in SEQ ID NO:60 (CDR1), SEQ ID NO:61 (CDR2) and SEQID NO:62 (CDR3) in a light chain of said antibody.
 2. A pharmaceuticalcomposition comprising as an active ingredient the isolatedpolynucleotide of claim 1 and a pharmaceutically acceptable carrier. 3.An isolated antibody polypeptide that specifically binds an exposedepitope shared by p53 mutant proteins and not by wild type p53 protein,wherein said polypeptide has affinity for said epitope of less than 25nanomolar, and wherein said polypeptide comprises the amino acidsequences set forth in SEQ ID NO:5 (CDR1), SEQ ID NO:6 (CDR2) and SEQ IDNO:7 (CDR3) in a heavy chain of said antibody and the amino acidsequences set forth in SEQ ID NO:45 (CDR1), SEQ ID NO:46 (CDR2) and SEQID NO:47 (CDR3) in a light chain of said antibody; or the amino acidsequences set forth in SEQ ID NO:5 (CDR1), SEQ ID NO:6 (CDR2) and SEQ IDNO:7 (CDR3) in a heavy chain of said antibody and the amino acidsequences set forth in SEQ ID NO:60 (CDR1), SEQ ID NO:61 (CDR2) and SEQID NO:62 (CDR3) in a light chain of said antibody.
 4. A pharmaceuticalcomposition comprising as an active ingredient the isolated antibodypolypeptide of claim 3, and a pharmaceutically acceptable carrier.
 5. Acomposition comprising a viral display vehicle comprising on the surfaceof the viral display vehicle the isolated polypeptide of claim
 3. 6. Apharmaceutical composition comprising as an active ingredient the viraldisplay vehicle of claim 5, and a pharmaceutically acceptable carrier.7. A nucleic acid construct comprising the isolated polynucleotide ofclaim 1, and a promoter for directing an expression of said isolatedpolynucleotide in cells.
 8. A method of inducing apoptosis and/or growtharrest of cancer cells, comprising contacting with or expressing in thecancer cells the isolated antibody polypeptide of claim 3, therebyinducing apoptosis and/or growth arrest of the cancer cells in vitro. 9.A method of inducing apoptosis and/or growth arrest of cancer cells,comprising contacting with the cancer cells the viral display vehicle ofclaim 5, thereby inducing apoptosis and/or growth arrest of the cancercells in vitro.
 10. A method of diagnosing a p53-related cancer in asubject comprising: (a) contacting a biological sample of the subjectwith the isolated antibody polypeptide of claim 3 under conditionssuitable for immunocomplex formation which comprises the isolatedantibody polypeptide and p53 mutant proteins; and (b) detectingformation of said immunocomplex, thereby diagnosing the cancer in thesubject.
 11. The isolated polypeptide of claim 3, wherein said epitopeis as set forth by SEQ ID NO:1.
 12. The isolated polypeptide of claim 3,wherein said antibody polypeptide is selected from the group consistingof a Fab fragment, an Fv fragment, a single chain antibody, a singledomain antibody and an antibody.
 13. The isolated polypeptide of claim12, wherein said single chain antibody is selected from the groupconsisting of SEQ ID NO:114 and SEQ ID NO:115.
 14. The nucleic acidconstruct of claim 7, further comprising an additional nucleic acidsequence encoding a nuclear localization signal (NLS) fused to saidpolypeptide.
 15. The nucleic acid construct of claim 14, wherein saidNLS is set forth by SEQ ID NO:134.
 16. The isolated polynucleotide ofclaim 1, wherein said polynucleotide further comprises an additionalnucleic acid sequence encoding a drug.
 17. The pharmaceuticalcomposition of claim 4, wherein said polypeptide further comprises anamino acid sequence of a drug.
 18. The pharmaceutical composition ofclaim 17, wherein said drug is a toxin and/or a chemotherapy drug. 19.The isolated polynucleotide of claim 1, wherein said polynucleotidefurther comprises an additional nucleic acid sequence encoding adetectable label.
 20. The isolated polypeptide of claim 3, wherein thepolypeptide further comprises a detectable label.
 21. The isolatedpolypeptide of claim 20, wherein said detectable label is biotin anddigoxigenin.
 22. The method of claim 10, wherein said biological sampleis selected from the group consisting of blood, lymph node biopsy, bonemarrow aspirate and a tissue sample.
 23. The isolated polypeptide ofclaim 12, wherein each of said Fab fragment, said Fv fragment, saidsingle chain antibody, said single domain antibody and said antibody ishumanized.
 24. A method of inducing apoptosis and/or growth arrest ofcancer cells, comprising contacting with or expressing in the cancercells an isolated antibody polypeptide that specifically binds anexposed epitope shared by p53 mutant proteins and not by wild type p53protein, wherein said polypeptide has affinity for said epitope of lessthan 25 nanomolar, thereby inducing apoptosis and/or growth arrest ofthe cancer cells in vitro.
 25. The method of claim 24, wherein saidantibody polypeptide is expressed on the surface of a viral displayvehicle.
 26. The method of claim 24, wherein said antibody polypeptidecomprises the amino acid sequences set forth in SEQ ID NO:5 (CDR1), SEQID NO:6 (CDR2) and SEQ ID NO:7 (CDR3) in a heavy chain of said antibodyand the amino acid sequences set forth in SEQ ID NO:39 (CDR1), SEQ IDNO:40 (CDR2) and SEQ ID NO:41 (CDR3) in a light chain of said antibody;the amino acid sequences set forth in SEQ ID NO:5 (CDR1), SEQ ID NO:6(CDR2) and SEQ ID NO:7 (CDR3) in a heavy chain of said antibody and theamino acid sequences set forth in SEQ ID NO:45 (CDR1), SEQ ID NO:46(CDR2) and SEQ ID NO:47 (CDR3) in a light chain of said antibody; or theamino acid sequences set forth in SEQ ID NO:5 (CDR1), SEQ ID NO:6 (CDR2)and SEQ ID NO:7 (CDR3) in a heavy chain of said antibody and the aminoacid sequences set forth in SEQ ID NO:60 (CDR1), SEQ ID NO:61 (CDR2) andSEQ ID NO:62 (CDR3) in a light chain of said antibody.